Báo cáo khoa học: The specific delivery of proteins to human liver cells by engineered bio-nanocapsules doc

In this study, the fusion of emerald green fluorescent protein EGFP at the C-terminus of the S region was designed to deliver proteins to human hepatocytes.. The dual C-terminal topologie

by engineered bio-nanocapsules

Dongwei Yu1, Chie Amano1, Takayuki Fukuda1,2, Tadanori Yamada3, Shun’ichi Kuroda3,4,

Katsuyuki Tanizawa3,4, Akihiko Kondo4,5, Masakazu Ueda4,6, Hidenori Yamada1, Hiroko Tada1 and Masaharu Seno1,4,7

1 Graduate School of Natural Science and Technology, Okayama University, Japan

2 Kobe R & D Center, Katayama Chemical Industries Co Ltd, Kobe, Japan

3 Institute of Scientific and Industrial Research, Osaka University, Japan

4 Research and Development Division, Beacle, Inc., Okayama, Japan

5 Faculty of Engineering, Kobe University, Japan

6 Keio University, School of Medicine, Tokyo, Japan

7 Research Center for Biomedical Engineering, Okayama University, Japan

A drug and gene delivery system has long been

consid-ered important for drug discovery and pharmaceutical

development In particular, the establishment of a

cell-or tissue-specific targeting method is the latest area of

focus Although, viral vectors, such as those utilizing

adenovirus or adeno-associated virus, have been devel-oped for gene therapy, the cell specificity must be ameliorated Some other problems, such as inflamma-tion, neutralizing antibodies, the dangers of mass pro-duction, and insertional mutagenesis, limit the use of

Keywords

bio-nanocapsule; L fusion protein; protein

delivery vector; specific infection; topology

Correspondence

M Seno, Graduate School of Natural

Science and Technology, Okayama

University, 3.1.1 Tsushima-Naka, Okayama

700-8530, Japan

Tel ⁄ Fax: +81 86 251 8216

E-mail: mseno@cc.okayama-u.ac.jp

(Received 20 March 2005, revised 9 May

2005, accepted 24 May 2005)

doi:10.1111/j.1742-4658.2005.04790.x

A bio-nanocapsule (BNC), composed of the surface antigen (sAg) of the hepatitis B virus, is an efficient nanomachine with which to accomplish the liver-specific delivery of genes and drugs Approximately 110 molecules of sAg are associated to form a BNC particle with an average diameter of

130 nm The L protein is an sAg peptide composed mainly of preS and S regions The preS region, with specific affinity for human hepatocytes, is localized in the N-terminus The S region following the preS has two trans-membrane regions responsible for the formation of particles In this study, the fusion of emerald green fluorescent protein (EGFP) at the C-terminus

of the S region was designed to deliver proteins to human hepatocytes Truncation of the C-terminus of the S region was required to obtain suffi-cient expression levels in Cos7 cells The nanoparticles that were produced delivered EGFP to human hepatoma cells, displaying the EGFP moiety outside, or enclosing it inside However, only a single orientation character-izes the particle, so that either type of L fusion particle could be effectively and independently separated by an antibody affinity column The dual C-terminal topologies of the L fusion particles designed in this study could

be applied to various proteins for the C-terminal moiety of the L fusion proteins, depending on the character of the proteins, such as cytoplasmic proteins, as well as cytokines or ligands to cell surface receptors We sug-gest that this fusion design is the most efficient way to prepare a BNC that delivers proteins to specific cells or tissues

Abbreviations

BNC, bio-nanocapsule; DDS, drug delivery system; DMEM, Dulbecco’s modified Eagle’s medium; EGFP, emerald green fluorescent protein;

ER, endoplasmic reticulum; FBS, fetal bovine serum; HBV, hepatitis B virus; IFN, interferon; PMSF, phenylmethanesulfonyl fluoride; sAg, surface antigen.

viral vectors in humans By contrast, liposomes as a

drug delivery vector have no limits to mass production

and do not have the drawbacks of viral vectors

How-ever, liposomes have a lower transfection efficiency and

cell⁄ tissue specificity compared with viral vectors, even

though they have the ability to transfer proteins into

cells To find a material that has good tranfection

effi-ciency free from the hazards of viral infection, we come

up with the idea of using a hollow particle derived from

recombinant viral envelope proteins The hepatitis B

virus (HBV) is a human liver-specific virus whose

3.2-kb genome codes three envelope proteins in a single

open reading frame (ORF) These encoded surface

anti-gens (sAgs) are called small (S), medium (M) and large

(L) proteins [1] In 1982, Valenzuela et al succeeded in

preparing recombinant sAg from yeast as S protein

particles with a diameter of 22 nm [2] Around 1990,

recombinant L proteins were also found to be

pro-duced in yeast cells as hollow virus-like particles and

were developed as immunogens for hepatitis B

vac-cines, which were proven safe for use in humans [3,4]

The recombinant L proteins formed a virus-like particle

with a diameter of approximately 200 nm when

pro-duced in Saccharomyces cerevisiae [3,5] We reported

that this particle was extremely useful as a vector to

target human hepatocyte in vivo exploiting the

charac-ter of infectivity of HBV limited to human liver [6]

Because this recombinant particle is empty, we named

this hollow nanoparticle a bio-nanocapsule (BNC) as it

represents an efficient nanomachine for achieving

liver-specific delivery of genes and drugs

Although electroporation is proposed as a

conveni-ent procedure for enclosing substances in the particle

[6], fusion to the C-terminus of L protein appears to

be the most efficient way to prepare a BNC to convey

foreign proteins Thus, fusing a suitable protein to the

tail should be a convenient way of preparing a BNC

that delivers proteins to human hepatocytes

Results

Evaluation of hepatitis B L fusion particles

The HBV L envelope protein is composed of three

major regions The preS1 region of 108 or 119 amino

acids at the N-terminus directly recognizes human

hepatocytes [1,7–9] The preS2 region of 55 amino acids

following the preS1 region has an affinity with

poly-merized albumin-mediated interactions The major 226

amino acids of the S region occupy the C-terminal half

of the L protein Current models for the

transmem-brane structure of the S region assume that both the

N- and C-termini are at external positions in mature

particles [10,11] Although it is predicted that four trans-membrane-like a helices are present in the S region, only the two in the N-terminus have been shown to be transmembrane helices [12–14] These two domains, which correspond to amino acids 8–22 and 80–98, respectively, are separated by a hydrophilic region that

is exposed to the internal side of the mature particle The C-terminal topology of the S region has not been challenged experimentally and remains to be investi-gated [12,15], whereas the preS region has been shown

to protrude into the lumen of the endoplasmic reti-culum (ER) [16] To enclose protein inside the BNC,

we designed emerald green fluorescent protein (EGFP) fusion at the C-terminus of the S region, with or with-out truncation, using 32, 45 and 54 amino acid residues from the C-terminus, respectively, to obtain four types

of L fusion particles, L-FLAG–EGFP, L(D32)-FLAG– EGFP, L(D45)-FLAG–EGFP and L(D54)-FLAG– EGFP, with an intervening sequence of FLAG-tag (Fig 1) Truncations were designed to shorten the C-terminus as much as possible for a single transmem-brane spanning region Because proline often functions

as a helix disruptor, the proline residues at 177 and 187 are considered in the truncation

According to the amino acid sequence, the mole-cular mass of L-FLAG–EGFP, which is the fusion protein between the full length of the L protein and EGFP, is calculated to be 68.5 kDa The apparent molecular mass of L-FLAG–EGFP is 71.5 kDa due to the glycosylated Asn146 in the S region on SDS⁄ PAGE [15,17] Taking the sizes of C-terminal truncation into account, the molecular mass of each L(D32)-FLAG–EGFP, L(D45)-FLAG–EGFP and L(D54)-FLAG–EGFP is calculated to be 68, 66.5 and 65.7 kDa, respectively

These L proteins fused to EGFP were transiently expressed in Cos7 cells (Fig 2A) During the culturing period, EGFP fluorescence was observed from the transfected Cos7 cells along the ER and Golgi, but not

in the nuclei, indicating that the fused protein was pro-duced in the secretional path Fusion proteins were detected with both anti-S and anti-GFP IgG in the cell lysates of transfected cells by western blotting at their expected sizes, as calculated (Fig 2B) Immuno-precipitates of the conditioned media, with either

anti-S, anti-FLAG or anti-GFP IgG, showed bands of fusion proteins, in addition to those in the cell lysates (Fig 2C) Secretion of L-FLAG–EGFP and L(D54)-FLAG–EGFP from Cos7 cells appeared limited, although it was, in fact, produced in sufficient quan-tity Expression and secretion of L(D32)-FLAG–EGFP were both low Secretion of L(D45)-FLAG–EGFP was obviously the highest of the four fusion constructs

The conditioned media were further assessed for the

secretion of these different constructs of particles using

enzyme immunoassay and fluorescence (Fig 3) When

the level of L-FLAG–EGFP was considered to be

100%, that of L(D45)-FLAG–EGFP was > 300% in

EIA The fluorescence from the EGFP moiety of

L fusion protein showed that L(D45)-FLAG–EGFP

was secreted at the highest ratio The analyses revealed

that L(D45)-FLAG–EGFP was suitably optimized for

production as the fusion protein

L(D45)-FLAG–EGFP particle formation was assessed

by sucrose gradient ultracentrifugation (Fig 4) When

cell extracts were subjected to ultracentrifugation, both

fluorescence and immunoreactivity were seen in the

same fraction at a density of 1.11 gÆmL)1, whereas the

native EGFP fluorescence peak was found to have a

density of 1.05 gÆmL)1 Peak fractions were analyzed by

western blotting to confirm the presence of fusion

pro-tein at 66.5 kDa The density of the particles obtained

from both cell extracts and the conditioned media was

equivalent to that of the BNC Whether from the

condi-tioned media or the cell extracts, L(D45)-FLAG–EGFP

has the potential to form a particle

Topology analysis of the C-terminus of L fusion

protein

Consisting of 56 amino acids, the primary structure of

the C-terminus of the S region is rich in hydrophobic

residues Therefore, the computer-assisted prediction

assumes that there should be two a helices, which traverse the ER membrane twice [11,18] In this con-text, EGFP fused to L protein with an inter-vening FLAG sequence may provide an excellent means of judging the C-terminal topology Because L-FLAG–EGFP could be immunoprecipitated by anti-FLAG and anti-GFP IgG (Fig 2C), the L pro-tein C-terminus should be located on the external side

of the particle This is consistent with results reported

by Eble et al [12]

Because generation of its fluorophore depends on the correct formation of a tertiary structure and can easily be detected by fluorescence microscopy, EGFP has quickly become a powerful research tool for gene expression and subcellular protein localization in liv-ing cells and organisms It was useful in this study, not only to estimate the level of L-EGFP fusion pro-tein expression, but also to observe the infection of particles by its fluorescence Within the structure of EGFP, chromophores are insulated by tightly woven barrel formations of b sheets, which offer strong resistance against proteolytic attack [19,20] This resistance was confirmed by incubation with protein-ase K at 60
C for 6 h (data not shown) As a result

of this resistance, a convenient protease protection assay was available to determine the topology of the fused protein, as described below However, slight digestion after 1 h of incubation was observed, indi-cating that the terminal sequences might be sensitive

to proteinase K (Fig 5)

Fig 1 Fusion proteins between L and EGFP The 720 bp egfp gene was ligated to the 3¢ side of the L gene of the hepatitis B virus flanking

a 39 bp FLAG as a spacer sequence in an open reading frame The resultant gene coding L-FLAG–EGFP was inserted downstream of a SRa promoter The C-terminus of the L protein was truncated by 32 (96 bp), 45 (135 bp) and 54 amino acid residues (162 bp) to optimize expres-sion of the L fuexpres-sion protein The resultant fuexpres-sion proteins are designated as L-FLAG–EGFP, L(n32)-FLAG–EGFP, L(n45)-FLAG–EGFP, and L(n54)-FLAG–EGFP TM1 and TM2 represent transmembrane regions of the L protein.

L(D45)-FLAG–EGFP particles were

immunoprecipi-tated with two different antibodies and treated with

proteinase K Western blots with anti-GFP IgG

showed two digested EGFP bands of I and II

(Fig 5A) when immunoprecipitated with anti-S IgG,

but only one band of II when immunoprecipitated

with anti-GFP IgG The only difference between these

two bands of I and II should be due to the size of

N-terminal sequence of EGFP protected from protein-ase K This result indicates that the C-terminus of the L-EGFP fusion protein may exhibit dual topology Because an anti-S IgG recognizes the immunodomi-nant a-epitope, which is common to all six genotypes

of HBV as the major surface region of the HBsAg envelope protein [21,22], this antibody will immuno-precipitate all of the L(D45)-FLAG–EGFP particles

By contrast, anti-GFP IgG selectively immunoprecipi-tate particles that display the EGFP moiety at the C-terminus on their surface With the immunoprecipi-tates, the EGFP moiety was processed to band II, the N-terminus of which was not protected from protein-ase K (Fig 5A) Anti-S IgG immunoprecipitates inclu-ded another topology, which protects the N-terminus

of the EGFP moiety from proteinase K There is no

A

B

C

Fig 2 Expression of L fusion proteins in Cos7 cells (A) Cells

were transfected with plasmids to express (a) EGFP, (b) L protein,

(c) L-FLAG–EGFP, (d) L(n32)-FLAG–EGFP, (e) L(n45)-FLAG–EGFP,

and (f) L(n54)-FLAG–EGFP, by electroporation Two days after

elec-troporation, the green fluorescence was observed under a confocal

microscope at a 63-fold magnification The bar scale shows 50 lm.

After 3 days, the Cos7 cells were harvested and disrupted by

sonica-tion (B) Cell lysates were probed in western blots with S or

anti-GFP IgG (C) Conditioned media were collected, immunoprecipitated

with anti-S, anti-GFP or anti-FLAG IgG, respectively, and subjected to

western blotting with anti-S IgG Lane 1, EGFP; lane 2, L protein; lane

3, L-FLAG–EGFP; lane 4, L(n32)-FLAG–EGFP; lane 5, L(n45)-FLAG–

EGFP; lane 6, L(n54)-FLAG–EGFP.

Fig 3 Secretion of L fusion particles evaluated by enzyme immuno-assay and fluorescence The HBsAg immunoreactivity (A) and the fluorescence (B) in the conditioned medium of the transfected Cos7 cells were measured and the percentages were calculated with the level of L-FLAG –EGFP expression assumed to be 100% n32, n45 and n54 denote L(n32)-FLAG–EGFP, L(n45)-FLAG–EGFP and L(n54)-FLAG–EGFP, respectively Each SD was calculated from three independent evaluations.

possibility that the dual C-terminal topologies coexist

in a particle However, there should be two types of

L(D45)-FLAG–EGFP particles, because EGFP

moiety-displaying particles immunoprecipitated with anti-GFP

IgG showed only single digested product band II If

the two types of C-terminal topology coexist in a parti-cle there should be two digested bands following immunoprecipitation by anti-GFP IgG, as revealed by the anti-S IgG Thus, we concluded that the EGFP moiety was protected from proteinase K by the mem-brane when it was located inside the particle, and that

it was slightly digested when located on the external side of a particle without membrane protection

To confirm this finding, we subtracted the L(D45)-FLAG–EGFP particles from the conditioned medium with anti-GFP IgG and protein G conjugated to agarose by immunoprecipitation The residual super-natant was subjected to repeated immunoprecipitation using the same procedure until the L fusion protein could not be detected by western blotting When the

A

B

C

Fig 4 Particle formation of L(n45)-FLAG–EGFP evaluated by

sucrose gradient ultracentrifugation (A) Cell extracts of the Cos7

cells transfected with L(n45)-FLAG–EGFP were subjected to

sucrose gradient ultracentrifugation and each fraction was

evalu-ated for immunoreactivity (e) and fluorescence (h) Native EGFP

was subjected to centrifugation and the fluorescence from EGFP

was simultaneously monitored (d) (B) Fractions that showed both

immunoreactivity and fluorescence in (A) were analyzed by western

blotting with anti-S IgG (C) L(n45)-FLAG–EGFP particles prepared

from conditioned medium (open triangle) and cell extracts [fraction

11 in (B); h] were compared with the BNC prepared from

recom-binant yeast [6] (s) for immunoreactivity The density of each

frac-tion was plotted by d.

A

B

Fig 5 Evaluation of the C-terminal orientation of the EGFP moiety in

L fusion particle (A) Two different L fusion particles were immuno-precipitated by either anti-S or anti-GFP IgG, and the immunoprecipi-tates were treated by proteinase K (pK) n32 and n45 denote L(n32)-FLAG–EGFP and L(n45)-FLAG–EGFP, respectively Digested products were detected with anti-GFP IgG The produced bands were indicated by arrows with I (28 kDa) and II (25 kDa) The native EGFP was treated with or without proteinase K and shown as the reference In (B) the conditioned medium containing L(n45)-FLAG– EGFP particles was immunoprecipitated by anti-GFP IgG (lane 1), and the process was repeated until no L fusion protein could be detected

by anti-S IgG (lane 2) The residual supernatant was immunoprecipi-tated by anti-S IgG (lane 3).

final subtracted fraction of the supernatant was further

immunoprecipitated with the anti-S IgG conjugated to

a microparticle, the L fusion protein band was still

detected in the fraction, thereby indicating the presence

of L fusion particles, which contained EGFP moieties

inside (Fig 5B)

Human hepatocyte specific delivery of EGFP

by infection

The preS1 peptide displayed on the surface of L

parti-cles recognizes the specific receptor present on human

hepatocytes and is essential for HBV infectivity [7,8]

This specific infectivity of the L particle should be

independent of the tolerable C-terminus truncation

Cell type-specific infection of L(D45)-FLAG–EGFP

particles was assessed on various human cancer cells

(Fig 6) After 9 h of incubation with L(D45)-FLAG–

EGFP particles, EGFP fluorescence was specifically

observed in human hepatocellular carcinoma HepG2

cells and NuE cells, whereas no EGFP fluorescence

was observed from either human colon adenocarcinoma

WiDr cells or human epidermoid carcinoma A431 cells

(Fig 6A) The EGFP fluorescence was not observed in HepG2 cells when incubated with BNC, EGFP or a mixture of BNC and EGFP (Fig 6B)

Discussion

In this study we attempted to establish a nanocapsule that efficiently delivers protein for tissue- or cell-type-specific targeting Based on our technology of BNC as

a delivery vector, we used a fusion strategy that ensures that the protein of interest is produced as a component of the particle The fusion proteins between

L protein and EGFP (L-FLAG–EGFP) were expressed with or without C-terminal truncation of L protein It was necessary to truncate the C-terminus of the L pro-tein by 45 amino acids for it to be efficiently secreted from the cells A EGFP moiety fused to the C-termi-nus of the L protein appears to prevent secretion, although the mechanism is not currently clear How-ever, incorrect folding of each half moiety of the fusion protein does not explain the low secretion, because we were able to prepare the particle from both conditioned media and cell extracts Once prepared,

Fig 6 Infection of L fusion particles in vitro (A) Five nanograms of L(n45)-FLAG-EGFP particles obtained from the conditioned medium were added to the culture media of 5 · 10 4 cells of HepG2 (a), NuE (b), WiDr (c) and A431 (d), respectively (B) Conditioned medium of non-transfected Cos7 cell (a), 5 ng of BNC from the conditioned medium (b), 100 ng of EGFP (c) and a mixture of 5 ng of BNC with 100 ng of EGFP (d), and 5 ng of L(n45)-FLAG-EGFP particles from the conditioned medium (e) were added to 5 · 10 4

cells of HepG2, respectively (C) Extracts of nontransfected Cos7 cells (a), 5 ng of L-FLAG-EGFP particles from the extracts of transfected Cos7 cells (b) and 5 ng L(n45)-FLAG-EGFP particles from the extracts of transfected Cos7 cells (c) were added to 5 · 10 4 cells of HepG2 The fluorescence was observed under a confocal microscope at a 63-fold magnification after 9 h infection Scale bar¼ 50 lm.

particles displayed immunoreactivity in an enzyme

immunoassay consistent with the fluorescence intensity,

when kept for 3 weeks at 4
C in the presence of

phe-nylmethanesulfonyl fluoride (PMSF) Furthermore, the

cell extract was able to directly infect HepG2 cells

(Fig 6C) These results indicate that the L-FLAG–

EGFP particle is stable, if properly prepared, and has

the potential to infect cells

Following optimization of the C-terminal truncation,

we attempted to determine the topology of the fused

protein in the particle because the purpose of this

study was to design a nanoparticle that incorporated a

foreign protein using a fusion strategy One of the

pur-poses of inserting a FLAG-tag between the C-terminus

of the L protein and EGFP was to study the topology

of the fused protein because we expected enterokinase

to specifically recognize and cleave FLAG peptide

Unexpectedly, this protease cleaved other basic

resi-dues in the L protein moiety, displaying many

degra-ded products, which confused us By contrast, the

strong resistance of EGFP to proteinase K was

extre-mely useful in studying the C-terminal topology of

L fusion protein Proteinase K treatment of the

L(D32)-FLAG–EGFP particles showed results similar

to those obtained with the L(D45)-FLAG–EGFP

parti-cle The hydrophobic sequence of approximately 20–30

amino acids in the C-terminus of the L(D32) or L(D45)

protein may traverse the membrane of lipid bilayer

However, our results show that this terminus is not

sufficiently hydrophobic to anchor the C-terminus in

the membrane, although it is sufficient to exhibit dual topology Based on the results of the proteinase K pro-tection assay, we proposed a model of the nanoparticle

of L fusion protein (Fig 7) We designated the parti-cle, whose N-terminal EGFP moiety was incorporated within the membrane, as type I, whereas type II denotes the EGFP moiety displayed on the surface of the particle Because the particle membrane protected the N-terminal part of the EGFP moiety, proteinase K digestion of type I showed the EGFP moiety to have a slightly higher molecular mass than that produced by the treatment of type II We scanned the western blots

in Fig 5A and analyzed the densities of the two bands

We found the ratio of band I to band II was 39 : 61

To confirm this ratio, we also compared the immuno-reactivity of the conditioned medium containing both type I and type II particles with that of the type II-subtracted medium by the anti-GFP IgG, as shown

in Fig 5B The ratio of the result is 100) 36, which means that the ratio of type I to type II is 36 : 64 These different procedures used to estimate the ratio

of the particles in two topologies lead to almost equal results Therefore, we concluded that nearly 40% of the particles are type I The secretion enhanced by C-terminal truncation might be explained by the fixed topology because we could not detect a particle with a mixed type I and type II topology in one particle However, it is difficult to find a determinant of the topology of the C-terminal moiety that causes it to be incorporated inside or displayed outside This unfixed

Fig 7 Proposed models of L fusion

particles Type I, EGFP incorporating

particle Type II, EGFP displaying particle.

pattern of topology might clarify the results of

previ-ous studies of the topology of the HBsAg protein It is

suggested that the C-terminus of the envelope protein

protrudes from the particle in 1987 [12] Localization

of HBV epitope by monoclonal antibodies revealed

that the residues 178–186 of the S peptide are exposed

on the surface of the virion particle [23] Kuroda et al

described that Asn146 was not glycosylated when the

recombinant L particle was prepared from yeast,

whereas it was glycosylated when expressed in

mam-malian cells [3] This suggests that this aspargine

resi-due is located at the border of the external region and

the membrane-bound region The C-terminal sequence

of 56 amino acids from 170 to 226 may be long

enough to traverse the membrane twice, although the

hydrophobicity is not sufficient to explain the topology

precisely It may be possible to design the C-terminal

region as the clear transmembrane region by replacing

it with one of the transmembrane-type receptors to

limit the topology of the particle to type I

Our BNC has the same tissue-specific infectivity as

HBV because of the N-terminal region of L protein,

preS1, which determines its narrow host range and

dis-tinct organ tropism The region from 3 to 77 amino

acid residues of preS1 is essential for this specificity

[24] To avoid impairing this selectivity, we fused

EGFP to the C-terminal of L protein, which was

suc-cessfully truncated in order to be secreted from

mam-malian cells and assembled to an L fusion particle

We previously reported that BNC containing DNA of

interest yielded a very high transfection efficiency with a

high specificity of gene transfer to human liver-derived

cells [6] The L fusion particle described here should

have equivalent specific transfection efficiency due to

the character of the preS1 region of the L protein The

advantage of the fusion particle is that there is no need

to incorporate proteins using specialized methods, such

as electroporation, for which it is difficult to establish

the efficiency needed to transfer genes and drugs into

cells Depending on the cell type, conditions vary and

optimization of the conditions may sometimes lead to a

10-fold increase in efficiency This was also the case with

our BNC, and we had to optimize the electroporation

conditions, depending on the substances to be

incorpor-ated An efficient procedure is required to eliminate

empty particles after electroporation in order to attain

the highest efficiency As for the L fusion particle, all of

the particles are destined to convey the protein of

inter-est with a transfection efficiency of nearly 100% directed

to human-derived liver cells

The L fusion particles designed in this study were

found to have dual C-terminal topologies, which could

easily be separated using antibodies There was no

dif-ference in specific infectivity among them when monit-ored using EGFP fluorescence (data not shown) This means that it is possible to choose various proteins for the C-terminal moiety of the L fusion proteins, depending on the character of the proteins to be fused This possibility will include cytoplasmic proteins, as well as cytokines or ligands, for the cell surface In this context, one of the candidates of the moiety might be interferon (IFN) which is used with ribavirin to treat hepatitis C virus-induced liver disease This therapy has many does-dependent side effects, such as depres-sion and insomnia The L–IFN fudepres-sion particle would

be extremely useful because it targets only the liver so that the dose administered could be low so that the side effects would not be a cause for concern

Retargeting of BNC by replacing the preS1 region with other targeting moieties or biorecognition mole-cules, such as ligands, receptors and antibodies as pre-viously proposed [6], should also be applicable to the

L fusion particles The greatest problem in using the particle is that people who have antibodies to HBV are increasing in number due to the widespread hepati-tis B vaccination program Stealth mutations at Gln129 and Gly145 to Arg would not only address this problem, but also lead to a design of the delivery vec-tor with extremely low immunogenicity [25,26] Thus,

we are developing our BNC for its potential to become

a practical vector of protein delivery

Experimental procedures

Cell cultures

Human hepatoma HepG2 cells, human squamous cell carci-noma A431 cells and human colon adenocarcicarci-noma WiDr cells were cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% (v⁄ v) fetal bovine serum (FBS; PAA Laboratories, Pasching, Austria) Human hepa-toma NuE cells were cultured in RPMI-1640 with 10% (v⁄ v) FBS African green monkey kidney-derived Cos7 cells for particle production were maintained in DMEM supple-mented with 5% (v⁄ v) FBS These cells were maintained at

37
C ⁄ 5%CO2

Construct of plasmids

The HBV L gene was excised from the plasmid pGLD LIIP39-RcT [7] and inserted into the XhoI site of pEGFP-N1 vector (Clontech, Mountain View, CA) Then the synthetic oligo-nucleotide coding FLAG-tag sequence (5¢-ATATATTGATTACAAGGATGACGACGATAAGA TA-3¢) was inserted between the AccI site close to the C-terminus of the L protein and the AgeI site at the

N-minus of EGFP in pEGFP-N1 The NotI site after the

ter-mination codon of EGFP was changed to the XhoI site

The resultant ORF of the L-FLAG–EGFP fusion protein

was excised with XhoI and inserted at the XhoI site

down-stream of the SRa promoter in the plasmid of pBO477,

which is a derivative of pTB1455 [27], to construct the

plasmid pBO572 We constructed three other expression

vectors pBO638, pBO637 and pBO822 for L proteins with

truncation by 32, 45 and 54 amino acid residues at the

C-terminus, respectively The resulting three types of L fusion

particles were designated L(D32)-FLAG–EGFP,

L(D45)-FLAG–EGFP and L(D54)-L(D45)-FLAG–EGFP (Fig 1)

Preparation of L fusion particles

Five micrograms of expression plasmid DNA were

trans-fected into 5· 106

of Cos7 cells by electroporation at

300 V⁄ 950 lF Transfected cells were first cultured for

14–16 h in 8 mL of DMEM containing 5% (v⁄ v) FBS in a

100 mm dish The medium was replaced with 8 mL of

CHO-S-SFM II (Invitrogen, Carlsbad, CA) and the cells

were cultured for a further 72 h The conditioned medium

was collected and condensed in a Vivaspin concentrator

tube (molecular mass cut-off at 100 kDa; Vivaspin,

Sarto-rius, Hannover, Germany) according to the manufacturer’s

instructions Cells were harvested by a cell scraper and

sus-pended in 100 lL of NaCl⁄ Piin DMEM per dish and then

sonicated for 30 s The supernatant from the cell extracts

was collected by centrifugation The concentration of L

fusion particles in the conditioned medium and in the cell

extracts was independently determined by IMx HBsAg

(Abbott Laboratories, Sligo, Ireland) The fluorescence of

EGFP from the particles was simultaneously measured by

an F-2000 fluorescence spectrophotometer (Hitachi, Tokyo,

Japan)

Sucrose gradient ultracentrifugation

L fusion particles were analyzed by sucrose gradient

ultra-centrifugation with himac CP70MX (Hitachi) as described

previously [3] Briefly, transfected cells were harvested by a

cell scraper in the NaCl⁄ Picontaining EDTA The wet cells

were suspended in buffer A [0.1 m sodium phosphate,

pH 6.8, 15 mm EDTA, 2 mm PMSF, 0.85% (w⁄ v) NaCl

and 1% (v⁄ v) Triton X-100], and then sonicated for 30 s

on ice Cell extracts in the supernatant were subjected to

sucrose gradient ultracentrifugation at 103 600 g for 14 h at

4
C in 27 mL of sucrose gradient of 10–50% (w ⁄ v) in

buffer A without Triton X-100 Fractions containing L

fusion particle were collected and dialyzed against buffer A

without Triton X-100 The dialyzed solution was again

sub-jected to sucrose gradient ultracentrifugation under the

same conditions The conditioned medium was also

subjec-ted to sucrose gradient ultracentrifugation after

condensa-tion with Vivaspin Each 1 mL, fraccondensa-tionated from the

top of the centrifugation tube, was analyzed for density, immunoreactivity (IMx) and fluorescence L fusion particle fractions were collected and dialyzed against NaCl⁄ Pi

for 16 h at 4
C The dialyzed solution was filtered through

a membrane filter (0.22 lm, MILLEX-HV, Millipore, Cork, Ireland) and stored at 4
C

Protease protection assay

The L fusion particles were immunoprecipitated with monoclonal anti-(S mouse epitope) IgG conjugated to microbeads contained in the IMx kit or with monoclonal anti-(GFP mouse epitope) IgG (Sigma, St Louis, MO) and protein G agarose (Invitrogen) The immunoprecipitates were washed five times with NaCl⁄ Pi, and resuspended in

10 lL of NaCl⁄ Pi Proteinase K (New England Biolabs, Beverly, MA) was then added to achieve a concentration of

100 lgÆmL)1 The suspension was incubated at 37
C for

1 h PMSF was added to 5 mm to stop the digestion

Transfection of L fusion particle

About 5· 104

cells of HepG2, NuE, WiDr and A431 were seeded in each well of a Laboratory-Tek chamber slide (Nunc, Naperville, IL) and cultured at 37
C in 5% (v ⁄ v)

CO2 After 12 h, the culture media were replaced with

300 lL of CHO-S-SFM II containing 5 ng of L(D45)-FLAG–EGFP particles, and the cells were cultured for a further 9 h The chambers were subsequently detached and the glass slide was washed with NaCl⁄ Pi The cells were covered with glass in the presence of NaCl⁄ Pi containing 10% (v⁄ v) glycerol, and the specific EGFP fluorescence was observed under a confocal microscope LSM 510 META (Zeiss, Jena, Germany)

Acknowledgements

The authors thank Mrs Kumiko Soga for her excellent technical assistance, and Mr Masayuki Kita for his continuous encouragement This project was supported

in part by Grants-in-Aid from the Ministry of Educa-tion, Culture, Sports, Science and Technology, Japan, and the Japan Science and Technology Corporation (Research Fund for Patenting)

References

1 Heermann KH, Goldmann U, Schwartz W, Seyffarth T, Baumgarten H & Gerlich WH (1984) Large surface pro-teins of hepatitis B virus containing the pre-S sequence

J Virol 52, 396–402

2 Valenzuela P, Medina A, Rutter WJ, Ammerer G & Hall

BD (1982) Synthesis and assembly of hepatitis B virus surface antigen particles in yeast Nature 298, 347–350

3 Kuroda S, Otaka S, Miyazaki T, Nakao M & Fujisawa Y

(1992) Hepatitis B virus envelope L protein particles,

synthesis and assembly in Saccharomyces cerevisiae,

purification and characterization J Biol Chem 267,

1953–1961

4 Fujisawa Y, Kuroda S, Van Eerd PM, Schellekens H &

Kakinuma A (1990) Protective efficacy of a novel

hepa-titis B vaccine consisting of M (pre-S2+S) protein

parti-cles (a third generation vaccine) Vaccine 8, 192–198

5 Yamada T, Iwabuki H, Kanno T, Tanaka H, Kawai T,

Fukuda H, Kondo A, Seno M, Tanizawa K & Kuroda S

(2001) Physicochemical and immunological

characteriza-tion of hepatitis B virus envelope particles exclusively

consisting of the entire L (pre-S1+pre-S2+S) protein

Vaccine 19, 3154–3163

6 Yamada T, Iwasaki Y, Tada H, Iwabuki H, Chuah MK,

VandenDriessche T, Fukuda H, Kondo A, Ueda M,

Seno M et al (2003) Nanoparticles for the delivery of

genes and drugs to human hepatocytes Nat Biotechnol

21, 885–890

7 Marion PL, Salazar FH, Alexander JJ & Robinson WS

(1979) Polypeptides of hepatitis B virus surface antigen

produced by a hepatoma cell line J Virol 32, 796–802

8 Neurath AR, Kent SB, Strick N & Parker K (1986)

Identification and chemical synthesis of a host cell

recep-tor binding site on hepatitis B virus Cell 46, 429–436

9 Neurath AR & Kent SB (1988) The pre-S region of

hepadnavirus envelope proteins Adv Virus Res 34,

65–142

10 Guerrero E, Gavilanes F & Peterson DL (1988) Model

for the Protein Arrangement in Hbsag Particles Based on

Physical and Chemical Studies, in Viral Hepatitis and

Liver Disease(Zuckerman AJ, ed.), pp 606–613 Alan

R Liss, New York

11 Strik HJ, Thornton JM & Howard CR (1992) A

topolo-gical model for hepatitis B surface antigen Intervirology

33, 148–158

12 Eble BE, MacRae DR, Lingappa VR & Ganem D

(1987) Multiple topogenic sequences determine the

transmembrane orientation of the hepatitis B surface

antigen Mol Cell Biol 7, 3591–3601

13 Bruss V & Ganem D (1991) Mutational analysis of

hepatitis B surface antigen particle assembly and

secre-tion J Virol 65, 3813–3820

14 Prange R, Nagel R & Streeck RE (1992) Deletions in

the hepatitis B virus small envelope protein: effect on

assembly and secretion of surface antigen particles

J Virol 66, 5832–5841

15 Bruss V, Gerhardt E, Vieluf K & Wunderlich G (1996)

Functions of the large hepatitis B virus surface protein

in viral particle morphagenesis Intervirology 39, 23–31

16 Eble BE, Lingappa VR & Ganem D (1990) The N-terminal (pre-S2) domain of a hepatitis B virus surface glycoprotein is translocated across membranes

by downstream signal sequences J Virol 64, 1414–1419

17 Sureau C, Fournier-Wirth C & Maurel P (2003) Role of

N glycosylation of hepatitis B virus envelope proteins in morphogenesis and infectivity of hepatitis delta virus

J Virol 77, 5519–5523

18 Berting A, Hahnen J, Kroger M & Gerlich WH (1995) Computer-aided studies on the spatial structure of the small hepatitis B surface protein Intervirology 38, 8–15

19 Yang F, Moss LG & Phillips GN Jr (1996) The molecu-lar structure of green fluorescent protein Nat Biotechnol

14, 1246–1251

20 Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY & Remington SJ (1996) Crystal structure of the Aequorea victoriagreen fluorescent protein Science 273, 1392– 1395

21 Kennedy RC, Ionescu-Matiu I, Adler-Storthz K, Henkel

RD, Sanchez Y & Dreesman GR (1983) Characteriza-tion of hepatitis B surface antigen monoclonal anti-bodies Intervirology 19, 176–180

22 Zuckerman JN & Zuckerman AJ (2003) Mutations of the surface protein of hepatitis B virus Antiviral Res 60, 75–78

23 Paulij WP, de Wit PL, Sunnen CM, van Roosmalen

MH, Petersen-van Ettekoven A, Cooreman MP & Heijtink RA (1999) Localization of a unique hepatitis B virus epitope sheds new light on the structure of hepati-tis B virus surface antigen J Gen Virol 80, 2121–2126

24 Le Seyec J, Chouteau P, Cannie I, Guguen-Guillouzo C

& Gripon P (1999) Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain J Virol 73, 2052–2057

25 Waters JA, Kennedy M, Voet P, Hauser P, Petre J, Carman W & Thomas HC (1992) Loss of the common

‘A’ determinant of hepatitis B surface antigen by a vac-cine-induced escape mutant J Clin Invest 90, 2543– 2547

26 Cooreman MP, van Roosmalen MH, te Morsche R, Sunnen CM, de Ven EM, Jansen JB, Tytgat GN,

de Wit PL & Paulij WP (1999) Characterization of the reactivity pattern of murine monoclonal antibodies against wild-type hepatitis B surface antigen to G145R and other naturally occurring ‘a’ loop escape mutations Hepatology 30, 1287–1292

27 Tada H, Kurokawa T, Seita T, Watanabe T & Iwasa S (1994) Expression and characterization of a chimeric bispecific antibody against fibrin and against urokinase-type plasminogen activator J Biotechnol 33, 157–174