A novel nonviral vector based on vesicular stomatitis virus

Journal of Controlled Release 68 (2000) 187–194 www.elsevier.com / locate / jconrel

A novel nonviral vector based on vesicular stomatitis virus Susumu Imazu a , Shinsaku Nakagawa a , Tsuyoshi Nakanishi a , Hiroyuki Mizuguchi b , Hidetoshi Uemura c , Osamu Yamada c , Tadanori Mayumi a , * a

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1 -6, Yamadaoka, Suita, Osaka 565 -0871, Japan b Division of Biological Chemistry and Biologicals, National Institute of Health Science, 1 -18 -1 Kamiyoga, Setagaya-ku, Tokyo 158 -8501, Japan c Research and Development Center, Fuso Pharmaceutical Industries, 2 -3 -30 Morinomiya, Joto-ku, Osaka 536 -0025, Japan Received 27 December 1999; accepted 24 March 2000

Abstract Here we report a simple and efficient method for nonviral gene transfer using liposomes which have envelope protein of vesicular stomatitis virus (VSV) on their surface (VSV–liposomes). We prepared VSV–liposome by fusing simple liposomes with VSV particles. The density of VSV–liposome fusion products was intermediated between that of liposomes and that of VSV particles. Furthermore, VSV–liposome fusion products included both viral proteins and lipids from liposomes, and were confirmed to be fusion products, but not adsorptive products, by the resonance energy transfer fusion assay. To evaluate whether these particles can efficiently introduce their internal contents into the cytoplasm of mammalian cells, we examined the delivery of fragment A of diphtheria toxin (DTA) by VSV–liposomes into the cytoplasm of FL cells. We found that VSV–liposomes encapsulating DTA were highly cytotoxic to the cells, while empty VSV–liposomes and plain liposomes encapsulating DTA were not, suggesting that VSV–liposomes delivered DTA into cytoplasm. Consistent with this, the cells cultured with plasmid DNA entrapped in VSV–liposomes and coding for firefly luciferase showed significant luciferase expression, whereas cells culture with plasmid DNA in plain liposomes and plasmid DNA–cationic liposomes complex did not. Thus, VSV–liposomes function as a simple and efficient nonviral vector for the delivery of DNA.  2000 Elsevier Science B.V. All rights reserved. Keywords: Nonviral vector; Liposome; Vesicular stomatitis virus; VSV–liposome; Fusion product

1. Introduction Various methods for introducing exogenous genes

Abbreviations: VSV, vesicular stomatitis virus; DTA, fragment A of diphtheria toxin; VSV–liposomes, vesicular stomatitis virus– liposome fusion products *Corresponding author. Tel.: 181-6-6879-8175; fax: 181-66879-8179. E-mail address: [email protected] (T. Mayumi).

into various types of cells in vitro and in vivo have been developed to study the biological functions of macromolecules, to establish animal models of human diseases, and for gene therapy. The vectors used in the clinical studies of gene therapy have recently been demonstrated by viral or nonviral methods. Gene delivery using virus vectors is generally effective because of utilizing viral infectivity. However, the structure and expression of genes are restricted by the character of virus genome, and the

0168-3659 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 00 )00250-9

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vectors have risks of appearance of replication competent virus. Moreover, for example, retrovirus vectors can not transfer the genes into nondividing cells and have risk of insertion mutation. Nonviral vectors for gene transfer, such as liposomes, are potentially safer than the viral vectors, but their efficiency of gene expression in vivo is much lower than that of viral vectors. The efficiency of liposomes as a gene transfer vector for in vivo use depends, to a large extent, on the ability of the recipient cells to internalize loaded liposomes into intracellular organelles as well as on the ability of the lysosomal enzymes to disrupt the liposome membrane, thereby releasing the content [1]. However, in many cases the intra lysosomal low pH environment and the various lysosomal enzymes present cause hydrolysis and inactivation of enclosed genes [2–4]. These processes result in a reduction in the utility of the loaded liposomes to serve as a gene transfer tool. Vesicular stomatitis virus (VSV) is a rhabdovirus containing one glycosylated protein, the G protein, which is vital for viral infectivity [5,6]. Infection of VSV virions into living cells requires two sequential steps. The first step involves the attachment of G protein to the phosphatidylserine on the cell surface. Following binding, virus internalization takes place via receptor-mediated endocytosis [7,8]. Subsequently, the intraendosomal low pH environment [9,10] induced fusion of the viral envelope with the membranes of endocytic vesicles. Finally, the VSV virion releases the viral nucleocapsid into the cytoplasm. The G protein is necessary for both the binding and fusion steps, as removal of the G protein by trypsinization [11–13] or reacting VSV with anti-G protein antibodies [14] results in a marked decrease in viral infectivity. Furthermore, the G protein is silent at neutral pH but exhibits fusion activity in the lower pH environment of the endosomal compartment, at about pH 6.1 [15]. This property of the G protein may be used to develop liposomes that provide efficient nonviral gene transfer. In previous studies, reconstituted membrane vesicles containing VSV G proteins isolated by detergent dialysis have been shown to fuse, similar to intact virus particles, with cell plasma membranes [16–18]. Due to the presence of viral fusion proteins, such vesicles avoid the degradation of their internal

contents by lysosomal enzymes, thus introducing their contents into the cells efficiently. However, entrapment of macromolecules in reconstituted virus envelopes is an inefficient process [19]. In the present study, we prepared VSV–liposomes fusion products (VSV–liposomes) by incubating simple liposomes with UV-inactivated VSV, followed by purification via sucrose density centrifugation. We demonstrated that VSV–liposomes could effectively mediate the transfer of their internal contents from loaded liposomes into living cells.

2. Materials and methods

2.1. Materials Egg phosphatidylcoline (PC) were purchased from Nippon Oil & Fats Co. (Tokyo, Japan). Cholesterol (Chol) was purchased from Sigma Chemical Co. (St. Louis, USA). L-a-phosphatidyl-L-serine solution from bovine spinal cord was purchased from Wako Pure Chemical (Osaka, Japan). N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine (N-Rh-PE) and N-(7-nitro-2,1,3-benzoxadiazol-4yl)-phosphatidylethanolamine (N-NBD-PE) were purchased from Avanti Polar Lipids, Inc (Alabama, USA). Fragment A of diphtheria toxin (DTA) was kindly provided by M. Nakanishi (Research Institute for Microbial Diseases, Osaka University, Osaka, Japan).

2.2. Cells FL cell lines, which are human epithelial-like cells derived from normal amniotic membrane, were kindly provided by National Institute of Infectious Diseases (Tokyo, Japan). They were grown in Eagle’s MEM supplemented with 5% fetal calf serum (FCS). LLCMK2 cells, which are monkey kidney cells, were kindly provided by M. Nakanishi. They were grown in Eagle’s MEM supplemented with 10% FCS, penicillin (100 Units / ml) and streptomycin (100 mg / ml).

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2.3. Virus VSV, New Jersey serotype, was kindly provided by National Institute of Animal Health (Tsukuba, Japan). VSV was grown in FL cells. After 1 day, supernatant was added to PEG 6000 and NaCl to the final concentration of 8% (W/ V) and 0.5 M, respectively, and then stirred gently at 48C for 3 h. The virus was concentrated by centrifugation at 48C at 15 0003g for 20 min and resuspended in NTE (0.13 M NaCl, 1 mM EDTA, 50 mM Tris–HCl, pH 7.8). The virus was purified by 0–60% stepwise sucrose density centrifugation at 48C for 45 min at 21 000 rpm in an SW28.1 rotor. Finally, the purified virus was pelleted by centrifugation, resuspended in NTE, and stored at 2808C. Genomic RNA of VSV was inactivated by ultraviolet light irradiation (40 J / m 2 ) just before preparation of VSV–liposomes.

2.4. Preparation of liposomes Unilamellar liposomes were prepared as described by Oku et al. [20] with some modifications. The liposomes were prepared from 12.7 mg lipids (PC / PS / Chol55:1:4 molar ratio). In some case, 0.2 or 1 mol% N-Rh-PE and 1 mol% N-NBD-PE was also added to the lipid mixture. A mixture of lipids in chloroform was subsequently dried down at 558C by the rotary evaporator to obtain a thin lipid on a glass vial. Liposome suspensions were prepared by dispersing the thin lipid film in 0.3 ml of balanced salt solution (BSS: 10 mM Tris–HCl, 150 mM sodium chloride, pH 7.6) containing 0.2 mg of DTA or 1.5 mg of luciferase expression plasmid DNA, pCAL2 [21], at 558C. The liposomes were frozen rapidly in liquid nitrogen and then left to thaw at 558C for 10 min. After three cycles of freezing and thawing, the liposomes were extruded through 0.1 mm pore polycarbonate filters. The VSV–liposomes were prepared by mixing liposomes with UV-inactivated VSV in citrate acid / NaOH buffer (140 mM NaCl, 2 mM MgCl 2 , 1 mM EGTA, 80 mM citrate acid, pH 5.5) on ice for 30 min and at 378C for 15 min with shaking. VSV–liposomes were purified from unbound virus and liposomes by a stepwise sucrose density gradient centrifugation (60, 45, 10 w / v%) at 48C for 1 h at 23 000 rpm (Beckman SW28.1). During centrifugation, the VSV–liposomes settled to

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the 10–45 w / v% interface. The VSV–liposome buffer was replaced with NTE by the ultracentrifugation at 48C for 1 h at 23 000 rpm. Liposome suspensions at 3.0 mg / ml contained 1.26 mg / ml DTA or 2.0 mg / ml pCAL2.

2.5. NBD/Rh method [22] Steady-state emission and excitation spectra were obtained using a Jasco spectrofluorometer equipped with crossed polarizers to reduce light scattering. The excitation and emission band slits were 1 mm wide. Peak absorbance of samples was kept to ,0.1 to reduce inner filter effects. The samples were excited at 460 nm, and the spectra of emission energy from 490 to 650 nm observed. Following each measurement, vesicles were disrupted with Triton X-100 (1% final concentration). This treatment eliminated energy transfer and allowed the determination of the concentrations of N-NBD-PE and N-Rh-PE from their emission intensities using direct excitation.

2.6. Cytotoxic activity in vitro To determine the cytotoxicity of VSV–liposomes containing DTA, 2.5310 4 FL cells seeded into each wells of 24-well plates were incubated with various concentrations of VSV–liposomes containing DTA at 378C for 3 h. After 21 h in culture, the cells were pulse-labeled with [ 35 S]methionine (8 mCi / ml, 3 h) and the [ 35 S] count incorporated into TCA-precipitable materials determined.

2.7. VSV–liposome mediated gene transfer Cells (5.0310 4 ) were seeded in 12-well plates and cultured with MEM supplemented with 10% FCS. On the following day, the cells were washed once with BSS and incubated with VSV–liposomes containing pCAL2 (0.2 mg / ml) suspended in MEM supplemented with 10% FCS. After 24, 48, 72, and 96 h, luciferase activity in the cells was determined using a luciferase assay system (PicaGene, Tokyo Inki Co. Ltd, Tokyo, Japan) and a luminometer (Lumat LB9501, EG & G Berthold, Bad Wildbad, Germany). Activity was recorded as relative light units (RLU) per mg protein.

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2.8. Plasmid DNA–cationic liposomes complex mediated gene transfer Lipofectin (GIBCO BRL, Gaithersburg, USA) was used as the cationic liposome for the transfection. Plasmid pCAL2 (0.2 mg) and Lipofectin (1.0 mg) in 100 ml of serum-free MEM were combined, mixed gently, and incubated at room temperature for 15 min. After the mixture was diluted with 0.8 ml of MEM supplemented with 12.5% FCS, the cells were processed using the same protocol as for the VSV– liposomes. DNA: Lipofectin ratio of 1:5 (w / w) was used, which was optimal conditions for gene transfer.

3. Results and discussion To efficiently prepare fusion products between VSV and liposomes, phosphatidylserine, a ligand of the VSV G protein, was included in the lipid

composition of the liposomes. The liposomes were incubated with VSV on ice for 30 min and at 378C for 15 min with shaking. The mixture was then subjected to stepwise sucrose gradient centrifugation (10, 45, 60%: 2, 6, 6 ml) in order to purify the VSV–liposome fusion products, and after centrifugation a total of 20 fractions collected 0.8 ml each from the bottom of the tube. The protein concentration of each fraction was measured to monitor the virus derived protein, while N-Rh-PE labeled liposomes were used to monitor the liposome-derived lipid. When VSV or liposomes alone were subjected to stepwise sucrose gradient centrifugation, virus was recovered from the bottom of the tube, corresponding to fractions 2–4, and liposomes from the top, corresponding to fractions 18–20 (Fig. 1), indicating that these particles had different densities. Thus, the VSV–liposome fusion products were likely to be of intermediate density. When a mixture of VSV and liposomes was subjected to stepwise suc-

Fig. 1. Fluorescent intensity of N-Rh-PE and protein concentration in each fraction after stepwise sucrose gradient centrifugation. The liposomes containing N-Rh-PE were incubated with UV-irradiated VSV at 48C for 30 min and then 378C for 15 min in citrate buffer (pH 5.5). The mixture was then subjected to stepwise sucrose density gradient centrifugation, and fluorescent intensity (A) and protein concentration (B) of each fraction determined. Top, middle, or bottom fraction corresponds to fractions 18–20, 9–11, or 2–4 in the figure, respectively.

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rose gradient centrifugation, particles were detected in not only top and bottom fractions but also in the middle fractions (fractions 9–11), which included both viral protein and lipid from liposomes (Fig. 1). This suggested that the particles in the middle fraction likely to be the product of fusion between virus particles and liposomes. These intermediate density particles were not observed when the virus was incubated with liposomes at 48C (data not shown). To further confirm that the particles in the middle fraction included the VSV–liposome fusion products but not adsorptive products, we prepared liposome containing N-NBD-PE and N-Rh-PE, and monitored fusion between VSV and liposomes using the resonance energy transfer fusion assay. When liposomes containing both N-NBD-PE and N-Rh-PE were excited at 470 nm, emission maxima at 530 and 585

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nm were observed. This latter peak, characteristic for N-Rh-PE, arose from fluorescence energy transfer between the donor and acceptor pair (Fig. 2A). In the case represented by detergent solubilization of the vesicles, the shoulder seen at this wavelength was due solely to the direct excitation of N-Rh-PE (Fig. 2C). In the case of the particles in the middle fraction, there was an increase in the fluorescence yield from N-NBD-PE (Fig. 2B). Together, these changes are indicative of reduction in the efficiency of energy transfer between N-NBD-PE and N-RhPE. VSV–liposomes lowered the surface density of the energy acceptor and, thus, decreased the efficiency of energy transfer compared to the starting liposomes containing N-NBD-PE and N-Rh-PE. Finally, as expected, VSV and liposomes containing both N-NBD-PE and N-Rh-PE mixture did not lead to change in the efficiency of energy transfer (data

Fig. 2. Effect of fusion between N-NBD-PE / N-Rh-PE liposomes and VSV on energy transfer. Liposomes containing 1 mol% N-NBD-PE and N-Rh-PE were prepared using the freezing and thawing method. The liposomes were incubated with UV-irradiated VSV at 48C for 30 min and then 378C for 15 min in citrate buffer (pH 5.5). VSV–liposomes were separated from unbound virus and liposomes by stepwise sucrose density gradient centrifugation. Various liposomes were equal to maximal fluorescence at 590 nm on excitation at 560 nm. Emission spectra were obtained by exciting the samples at 460 nm. (A) Liposome, (B) VSV–liposome, (C) VSV–liposome detergent.

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not shown). Therefore, the particles in the middle fraction were indeed VSV–liposome fusion products. To evaluate whether VSV–liposome fusion products can efficiently introduce their internal contents into the cytoplasm of mammalian cells, we prepared VSV–liposomes encapsulating DTA and examined subsequent inhibition of protein synthesis in FL cells in vitro. DTA is known to kill cells by inactivating elongation factor 2, even when only a few molecules of this protein are introduced into the cytoplasm [23]. In contrast, DTA is completely non-toxic if independently taken up by endocytosis, as it cannot reach the cytoplasm due to degradation by lysosomal enzymes. Therefore, DTA is a superior marker protein to evaluate whether drug carriers can deliver their load into the cytoplasm. As shown in Fig. 3, DTA in VSV–liposomes exhibited high level of inhibition of the protein synthesis in FL cells, but

neither DTA in plain liposomes nor empty VSV– liposomes had any influence on the protein synthesis. Next, we examined the efficiency of expression of genes encapsulated in VSV–liposomes. To this end we used plasmid pCAL2, which contains the luciferase gene driven by the cytomegalovirus enhancer / chicken b-actin hybrid promoter. The results in Table 1 show luciferase activity in LLCMK2 cells transfected with various formulations of pCAL2 in the presence of FCS. VSV–liposomes (0.2 mg of pCAL2 / ml) exhibited marked luciferase expression in LLCMK2 cells, and VSV–liposomes were required 1 day more long than cationic liposomes for the highest expression of activity. In contrast, little luciferase activity was observed when cells incubated with plain liposomes containing 10 mg of pCAL2 / ml. In addition, cells incubated with pCAL2 (1.0 mg / ml)–cationic liposomes (lipofectin) complex, a

Fig. 3. Effect of VSV–liposomes containing DTA on FL cells. FL cells were incubated with various concentrations of empty liposomes (^), liposomes containing DTA (m), empty VSV–liposomes (s), VSV–liposomes containing DTA (d) at 378C for 3 h and cultured with normal medium for 24 h. The cells were then pulse-labeled with [ 35 S]methionine for 3 h, and the [ 35 S] counts incorporated into TCA-precipitable materials determined.

S. Imazu et al. / Journal of Controlled Release 68 (2000) 187 – 194 Table 1 Effect of duration of culture on luciferase activity in LLCMK2 cells transfected by VSV–liposome of lipofectin Vector

VSV–liposome Lipofectin Liposome b a b

Luciferase activity (RLU / mg protein)a Day 1

Day 2

Day 3

Day 4

255680 765 1268

590647 1062 562

2127680 861 161

1340629 561 161

Results are mean6S.D. for triplicate. Liposomes (10 mg pCAL2 / ml) were transfected to LLCMK2.

common nonviral gene transfer vector, did not appear to express luciferase (Table 1). The sensitivity of DNA–cationic liposomes complex mediated-gene transfer to serum proteins may be due to either instability of the complex in serum, as DNA is degraded by nucleases in serum, or the cationic liposomes themselves being unstable in the serum. In contrast, the DNA molecules in the VSV– liposomes were encapsulated in the membrane structure and protected from nucleases, while the VSV– liposomes themselves were stable in the serum. In fact, almost all DNA molecules that formed complexes with cationic liposomes were broken down when they incubated in 10% rat serum for 0.5 h, while almost all DNA entrapped in the VSV–liposomes (more than 90% of DNA) remained intact, even when they incubated in 50% of rat serum for 1.5 h (data not shown). Numerous studies have demonstrated that various enveloped viruses, such human immunodeficiency virus type 1 [24], influenza virus [25], Sendai virus [26,27] or Semliki Forest virus [28] are able to fuse with liposomes. Previously, we reported that Sendai virus–liposome fusion products could introduce their internal contents into the cytoplasm of mammalian cells in a receptor-dependent manner similar to the native virus [21,29–31]. However, Sendai virus fused with erythrocyte, and instability into the plasma. Sendai virus–liposome fusion products were caused to hemolysis and broken down immediately into the plasma. Therefore the way of administration of them was restricted to local administration or perfusion of organs. In the present study, we showed that VSV–liposomes are capable of delivering plasmid DNA into the cytosol and thereby inducing significant gene expression. VSV–liposomes did not

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have hemolysis activity against human erythrocyte, and were stabilized relatively into the plasma. We speculate that these characteristics of VSV– liposomes-mediated gene transfer can be attributed to those of VSV. So VSV–liposomes will be more widely applicable than Sendai virus–liposome fusion products in vivo. These observations indicate that virus–liposomes fusion products may function as an efficient tool with which to deliver various agents into target cells. In conclusion, we have demonstrated here that VSV can also mediate the transfer of both proteinaceous agents and plasmid DNA from loaded liposomes into living cells. The present results will provide a useful base for the design of nonviral liposomal vectors.

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