Development of a virus-mimicking nanocarrier for drug delivery systems: The bio-nanocapsule

    Development of a Virus-mimicking Nanocarrier for Drug Delivery Systems: the Bio-nanocapsule Masaharu Somiya, Shun’ichi Kuroda PII: DOI: Reference:

S0169-409X(15)00228-8 doi: 10.1016/j.addr.2015.10.003 ADR 12845

To appear in:

Advanced Drug Delivery Reviews

Received date: Revised date: Accepted date:

13 June 2015 21 September 2015 9 October 2015

Please cite this article as: Masaharu Somiya, Shun’ichi Kuroda, Development of a Virusmimicking Nanocarrier for Drug Delivery Systems: the Bio-nanocapsule, Advanced Drug Delivery Reviews (2015), doi: 10.1016/j.addr.2015.10.003

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For Advanced Drug Delivery Reviews

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Development of a Virus-mimicking Nanocarrier for Drug Delivery Systems: the

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The Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047,

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Japan b

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601,

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Japan

*Correspondence to:

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Japan Society for the Promotion of Science, Tokyo 102-0083, Japan

Prof. Shun’ichi Kuroda, Ph.D.

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c

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Masaharu Somiyaa,b,c and Shun’ichi Kurodaa,b,*

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Bio-nanocapsule

The Institute of Scientific and Industrial Research

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Osaka University Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan Tel.: +81-6-6879-8460

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Fax: +81-6-6879-8464 E-mail: [email protected]

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Abstract

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As drug delivery systems, nanocarriers should be capable of executing the

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following functions: evasion of the host immune system, targeting to the diseased site,

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entering cells, escaping from endosomes, and releasing payloads into the cytoplasm. Since viruses perform some or all of these functions, they are considered naturally occurring nanocarriers. To achieve biomimicry of the hepatitis B virus (HBV), we generated the ―bio-nanocapsule‖ (BNC)—which deploys the human

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hepatocyte-targeting domain, fusogenic domain, and polymerized-albumin receptor domain of HBV envelope L protein on its surface—by overexpressing the L protein in

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yeast cells. BNCs are capable of delivering various payloads to the cytoplasm of human hepatic cells specifically in vivo, which is achieved via formation of complexes with various materials (e.g., drugs, nucleic acids, and proteins) by electroporation, fusion

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with liposomes, or chemical modification. In this review, we describe BNC-related

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technology, discuss retargeting strategies for BNCs, and outline other virus-inspired

Keywords:

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nanocarriers.

Active targeting, biomimicking, hepatitis B virus, liposome, membrane fusion,

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virus-like particle

Abbreviations:

BAP, biotin-acceptor peptide; BNC, bio-nanocapsule; CPP, cell penetrating peptide; EGFP, enhanced green fluorescence protein; EGE, epidermal growth factor; EGFR, epidermal growth factor receptor; EPR, enhanced permeability and retention; DDS, drug delivery system; HBV, hepatitis B virus; HSA, human serum albumin; HSPG, heparin sulfate proteoglycan; LP, liposome; NHS, N-hydroxysuccinimide; NTCP, sodium taurocholate cotransporting polypeptide; PAR, polymerized-albumin receptor; PEG, polyethylene glycol; PEI, polyethyleneimine; RES, reticuloendothelial system; VLP, virus-like particle.

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1.

Introduction

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The bio-nanocapsule (BNC)

BNC: a recombinant hepatitis B virus (HBV) envelope L protein

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2.1.

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Table of contents

2.2.

The BNC as a human hepatocyte-targeting nanocarrier

2.3.

Pleiotropic functions of the BNC

Human hepatic cell-specific targeting activity

2.3.2.

Fusogenic activity

2.3.3.

Stealth activity

2.3.4.

Self-organizing activity

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Methods for incorporation of payloads into BNCs Electroporation

2.4.2.

Genetic fusion

2.4.3.

Chemical conjugation

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2.4.1.

2.4.4.

2.4.6. 2.5.

Liposome fusion Virosome formation

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2.4.5.

Polymer fusion

Retargeting of BNCs for various targets Modification of BNCs for efficient intracellular delivery

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2.6.

Virus-inspired nanocarriers 3.1.

4.

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2.3.1.

2.4.

3.

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particle

Virus-like particles

3.2.

Virosomes

3.3.

Virus-mimicking nanocarriers

Conclusion

Acknowledgements References

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1. Introduction

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Targeted drug delivery systems (DDSs), which deliver medication to diseased

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sites in patients, are recognized as key technologies for the effective treatment of

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disease with fewer side effects [1]. With the advent and progress of nanotechnologies, various nanoparticles have been developed and applied clinically as DDS nanocarriers [2,3]. A notable example is Doxil, liposomal doxorubicin, which is the most successful nanomedicine in production and has already been commercialized for the treatment of

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breast cancer [4,5]. Nanomicelles that encapsulate anti-cancer drugs (e.g., paclitaxel and cisplatin) are another promising nanomedicine currently applied to treat cancer in

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ongoing clinical trials [6]. These nanomedicines are usually modified with polyethylene glycol (PEG) chains on their surface, which facilitates their evasion from the non-specific adsorption of opsonins and capture by macrophages of the

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reticuloendothelial system (RES) [7]. Furthermore, owing to their approximate 100-nm

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size, they can circumvent the glomerular filtration process, which filters only small molecules [8]. These properties contribute to prolongation of the nanomedicines’ blood

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clearance time. Since tumors that show active angiogenesis have relatively leaky vasculature, ~100-nm-sized nanomedicines are extravasated from vessels and can accumulate in tumors (i.e., passive targeting via the enhanced permeability and retention

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(EPR) effect) [9–11]. Thus, passive targeting-based nanomedicines could provide substantial anti-tumor efficacy with fewer side effects. However, the immature vasculature and interstitium found in some tumors can hamper the delivery of sufficient anti-cancer drugs [12,13]. Therefore, it is widely recognized that the next generation of nanomedicines should harbor, in addition to passive targeting, active targeting machinery based on biomolecular recognition (e.g., ligand-receptors, antigen-antibodies, and sugar chain-lectins). As tumor-targeting biomolecules, transferrin [14] and folate [15] have been examined in numerous studies, and their receptors are abundantly expressed in tumors. Transferrin-conjugated cyclodextrin-based nanoparticles have been developed as a nanocarrier for tumor-specific siRNA (short interfering RNA) [16], with RNA interference now successfully demonstrated in humans [17]. Folate has also been conjugated with nanoparticles, and the effectiveness of these conjugates as anti-tumor 4

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drugs or imaging agents has been examined in preclinical studies [15]. In addition, antibodies recognizing tumor-specific surface antigens are frequently used as

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tumor-targeting nanocarriers [18]. These tumor-specific nanocarriers can bind to the

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surface of tumor cells, enter into cells via endocytosis, and move to endosomes and/or

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lysosomes. To avoid the degradation of drugs in lysosomes, nanocarriers should escape from endosomes/lysosomes and then release their payloads in the cytoplasm. Cytoplasmic delivery is particularly crucial for the most promising therapeutic agents, siRNAs, which exhibit their biological function in the cytoplasm [19]. Furthermore,

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active targeting-based nanomedicines are expected to exhibit therapeutic effects in various diseases, including those that are unrelated to tumors and do not always show

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the EPR effect. Considering all the evidence, the ideal nanocarrier should be capable of executing the following functions concurrently: (a) evasion of the host immune system (stealth activity), (b) targeting to the diseased site (active targeting), (c) entering cells,

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(d) escaping from endosomes/lysosomes to the cytoplasm, and (e) releasing payloads.

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However, assembling a nanocarrier with these capabilities into the limited space of a single nanostructure has not yet to be accomplished because of the currently inadequate

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techniques available for nanofabrication of biomolecules. Some viruses are known to infect specific tissues and organs in the human body [20]. Following their invasion of the body via various routes, viruses circulate

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through body fluids, expiration, and gastrointestinal content; they reach target tissues and organs by repelling the host immune system; and, finally, they enter the cells. Once in the cell, viruses deliver their genetic material with the help of intracellular transport systems. The extra/intracellular trafficking of viruses demonstrates that they possess the necessary abilities required to make them ideal nanocarriers. Specifically, viruses can be considered natural-occurring nanocarriers; thus, we have been inspired to develop novel nanocarriers that are biomimics of viruses.

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2. The bio-nanocapsule (BNC) BNC: a recombinant hepatitis B virus (HBV) envelope L protein particle

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2.1.

HBV is known to infect human liver cells specifically and cause severe liver

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diseases, such as hepatitis, cirrhosis, and liver cancer [21]. The HBV virion, namely the ~42-nm-sized spherical Dane particle, is composed of three types of envelope protein (small [S], middle [M; pre-S2+S], and large [L; pre-S1+pre-S2+S] proteins; also

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denoted as HBsAg [HBV surface antigen]), a lipid bilayer, core proteins (HBcAg, HBV core antigen), DNA polymerase, and genomic DNA (Fig. 1, left). Since approximately

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350 million people are chronically infected with HBV worldwide, vaccination is considered the most effective solution for reducing the occurrence of HBV-related diseases. The first generation HB vaccine, which consisted mainly of S protein particles,

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was produced from the plasma of non-active HB patients. In the 1980s, due to the

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limited availability of plasma from HB patients, S protein was expressed as particles in recombinant eukaryotic cells (e.g., yeast cells, CHO cells) and formulated into the

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second generation HB vaccine [22]. However, the second-generation HB vaccine was not fully immunogenic. To enhance its immunogenicity, pre-S2 region-appended S protein (M protein) was expressed as particles in yeast cells and used as an immunogen

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for the third generation HB vaccine [23]. This vaccine elicited effective levels of anti-pre-S2 and anti-S antibodies in clinical trials [24]. Subsequently, several researchers have attempted to synthesize pre-S1+pre-S2 region-appended S protein (L protein) particles, but the synthesis of L protein was unexpectedly inhibited by the involvement of the pre-S1 region. In 1992, we found that the addition of an N-terminal signal peptide could overcome the inhibition of L protein synthesis, and for the first time we accomplished overexpression of L particles in yeast cells [25]. Following the disruption of yeast cells with glass beads, the L particles were readily purified by using a process of heat-treatment, affinity column chromatography, and gel filtration [26]. L particles are ~100-nm-sized spherical hollow particles that consist of a liposome (LP) embedded with approximately 110 L protein molecules [27](see Fig. 4A). Anti-pre-S1, anti-pre-S2, and anti-S antibodies can recognize L particles efficiently, which indicates that the N-terminal half of the L protein (the pre-S1+pre-S2 region) is deployed 6

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outwardly on the surface of the L particle, similar to HBV. This similarity in surface structure prompted us to consider the L particle as a biomimic of HBV; in further

The BNC as a human hepatocyte-targeting nanocarrier

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2.2.

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studies, we designated this biomimic as a ―bio-nanocapsule‖ (BNC) (Fig. 1, right).

Nearly half a century has passed since the discovery of the HBV virion [28], but the molecular basis of the infection mechanism of HBV has not yet been fully

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elucidated [29]. In 1986, Neurath et al. [30] demonstrated that the human hepatocyte-specific recognition domain resides at 10–36 aa of the pre-S1 region

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(hereafter, the amino acid residue numbers are based on BNCs). They also showed that either anti-pre-S1 antibodies or a pre-S1 peptide vaccine could protect chimpanzees from HBV infection [31]. These data suggested that BNCs (containing L proteins

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exclusively) could recognize human hepatic cells specifically in vivo. In 2003,

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following the incorporation of fluorescent dye or an EGFP (enhanced green fluorescence protein)-expression plasmid by electroporation, the delivery of payloads to

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human hepatic cells by BNCs was confirmed in vitro and in vivo (through intravenous administration) [32]. When the expression plasmid for blood clotting factor IX was incorporated, the intravenously injected BNCs maintained the factor IX concentration in

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mice at levels above those sufficient for treatment of medium hemophilia B, and did so for nearly 1 month. After BNCs containing the HSV-tk (herpes simplex virus 1-derived thymidine kinase) gene were administrated intravenously, the tumor growth of a xenograft mouse model was synergistically suppressed by subcutaneously injected ganciclovir [33]. Furthermore, in SCID mice harboring human normal liver tissues under their kidney skin, BNCs containing fluorescent dye accumulated in the human liver tissues specifically, rather than in the native mouse tissues, following intravenous injection [34]. These results indicated that BNCs could deliver substantial amounts of various payloads (e.g., fluorophores and genes) through intravenous injection, not only to human hepatic cancer-derived tumors but also to human normal liver tissues in vivo, presumably by biomimicking the infection machinery of HBV.

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2.3.

Pleiotropic functions of the BNC

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The BNC has a simple structure consisting of L protein and a lipid bilayer

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exclusively. All of the functions prerequisite for in vivo human hepatic cell-specific

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delivery can be attributed to the ectodomain of L protein, namely the pre-S1 region, pre-S2 region, and N-terminal part of the S region (Fig. 2).

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2.3.1. Human hepatic cell-specific targeting activity

Recent studies on HBV infection machinery strongly suggest that HBV

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interacts initially with ―low-affinity receptors‖ and then switches to ―high-affinity receptors‖ for the commencement of endocytic internalization [29]. Heparin sulfate proteoglycan (HSPG), which is abundantly expressed in the extracellular matrix, was

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identified as a low-affinity receptor because heparin inhibits the HBV infection [35,36].

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HSPG is also expressed in other (not hepatic) cells and acts as a receptor for initial attachment of other viruses [37]; thus, the binding of HSPG with HBV is necessary for

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HBV infection but not essential for the determination of HBV tropism. Recently, sodium taurocholate cotransporting polypeptide (NTCP), a liver-specific bile acid transporter, has been identified as one of the functional receptors of HBV [38]. Ectopic

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NTCP expression permits HBV infection in non-susceptible cells [39–41]. NTCP binds to the myristoylated form of pre-S1 peptide (2–48 aa) with higher affinity rather than binding to the intact form [42]. Thus, NTCP is now recognized as one of the high-affinity HBV receptors. The human hepatic cell-specific infection machinery of BNCs is considered comparable to that of HBV [43]. BNCs were also shown to bind to low-affinity receptors and subsequently high-affinity receptors, and then internalize via the endocytic cascade [43]. Since heparin efficiently inhibits the binding of BNCs to human hepatic cells [44], HSPG might act as a low-affinity receptor for BNCs. In a model of HBV, BNCs could bind and internalize to human hepatic cells specifically at the same rate as HB patient-derived HBsAg [43], which leads us to suggest that NTCP could interact with BNCs (as well as HBV) as a high-affinity receptor. However, BNCs lack the myristoyl group because of the yeast expression system used for their production. 8

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Additionally, we recently observed that overexpressed NTCP did not significantly contribute to BNC binding in cells (unpublished data). Based on existing evidence,

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other human liver-specific factors, in addition to NTCP, could be involved in the

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binding and internalization of BNCs to human hepatic cells, and these may act as

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high-affinity receptors for BNCs and participate in the early infection machinery of HBV (Fig. 2).

Fusogenic activity

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2.3.2.

Similar to other envelope viruses (e.g., human immunodeficiency virus-1

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[HIV-1], influenza virus, and Sendai virus) [45], HBV also attaches to target cells via the interaction with specific receptors, is then promptly internalized into cells via endocytosis, and ultimately escapes from endosomes/lysosomes through membrane

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fusion between the endosomal membrane and HBV envelope membrane [46].

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Fusogenic activity of HBV envelope protein is a prerequisite for the initiation of this fusion event. Although fusogenic domains have already been identified at the center of

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the pre-S2 region (149–160 aa) [47] and the N-terminal part of the S region (164–186 aa) [48] (Fig. 2), the contribution of these domains to the fusion event of HBV is currently unclear. Recently, in a lipid-mixing assay with LPs and BNCs, we observed

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that the N-terminal peptide (9–24 aa) of the pre-S1 region possesses strong fusogenic activity [49](Fig. 2). When pre-S1-deleted BNC or anti-pre-S1 antibody was added to the assay, the fusogenic activity of BNCs was completely abolished; this indicates that the fusogenic domain of the pre-S1 region could exclusively start the fusion event. Furthermore, the fusogenic activity was increased in a low pH-dependent manner (below pH 5.5), which strongly suggests that the envelope membrane of the BNC (as well as that of HBV) fused with the endosomal membrane under the low pH conditions of late endosomes/lysosomes through this fusogenic domain [49]. When LPs (as a model of endosomes) containing fluorophore and quencher were incubated with BNCs at pH 4.5, the fluorophores were promptly released from LPs. Additively, BNCs containing doxorubicin were incubated with LPs at pH4.5, the drug was also promptly released from BNCs [49]. Considering these findings, the fusogenic domain of the pre-S1 region may play a crucial role in the disruption of endosomal membrane and 9

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BNC membrane in acidic condition, leading to the endosomal escape of the content of BNCs (probably as well as an uncoated form of HBV). This model agreed well with the

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early infection machinery proposed for HBV [29], which supports the endosomal escape

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of uncoated HBV.

2.3.3. Stealth activity

When nano-sized materials are injected into the blood, the RES readily

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captures them; however, some viruses can evade the host immune system (i.e., they show stealth activity). As demonstrated in SCID mice harboring human normal liver

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tissue under their kidney skin, intravenously injected BNCs accumulate specifically to human liver tissues and not RES-rich organs [34]. BNCs, and presumably HBV, are postulated to possess stealth activity. HBV is known to associate with monomeric

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human serum albumin (HSA), and more preferentially to polymerized-HSA [50,51],

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through the polymerized-albumin receptor (PAR) domain of the pre-S2 region (120–129 aa) [52] (Fig. 2). Since albumin is the most abundant protein in the blood, HBV could

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evade adsorption from opsonins (e.g., antibodies and complements) by recruiting albumins on its surface. Indeed, we have observed that the synthetic peptide encompassing the PAR domain can bind to polymerized-HSA in vitro, and that the

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nanoparticles displaying the peptide evade phagocytosis by Kupffer cells, which are responsible for the elimination of exogenous particles in the liver (unpublished data). Therefore, the binding activity of BNC to HSA in the pre-S2 region may contribute to the evasive abilities of the nanocarrier. In conventional nanomedicines, PEG is used to modify surfaces for the acquisition of stealth activity; however, the induction of anti-PEG IgM antibodies often impeded repetitive administration of PEGylated nanomedicines (i.e., the accelerated blood clearance phenomenon [53]). The albumin-coating strategy, inspired by HBV, could be an alternative method for extending the blood clearance time of nanoparticles.

2.3.4. Self-organizing activity

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When L proteins are synthesized in yeast cells, they initially accumulate in the endoplasmic reticulum (ER) as transmembrane proteins; subsequently, they recognize

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each other, spontaneously aggregate into particle structures in a budding manner, and

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then move to the ER lumen [25]. Owing to the self-organizing activity in the S region,

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the L proteins can form ~100-nm-sized spherical hollow particles concurrently with outward deployment of the pre-S1+pre-S2 region. At least three transmembrane domains within the S region are indispensable for the formation of the L particle (Fig. 2). Of these, α-helical structures are maintained by inner-/outer-disulfide bonds.

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Furthermore, a mutational study demonstrated that 6 out of 14 Cys residues in the S region are essential for particle formation [54]. The envelope structure of BNC allowed

Methods for incorporation of payloads into BNCs

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2.4.

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us to change the diameter of BNC itself from ~100 nm to ~30 nm by using extruder.

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HBV harbors its genetic material, used to establish infection in human liver cells, in its inner space. For human liver-specific delivery of payloads, BNCs must also

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incorporate their payloads (e.g., drugs, nucleic acids, or proteins) in their hollow inner space. Alternatively, the L protein can be genetically fused with payloads, the surface of the BNC can be modified with payloads, or the BNC can be fused with other

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nanocarriers containing payloads. In this section, we describe the methods employed to incorporate the various BNC payloads (Fig. 3; Table 1).

2.4.1.

Electroporation

The BNC is composed of 90 wt% glycoproteins and 10 wt% phospholipids [55]; therefore, we postulated that electroporation, which is widely used for the transfection of various cells [56], could transiently generate pores across the BNC membrane. Indeed, BNCs can incorporate fluorophores and plasmids (up to ~40 kilobase pairs) by electroporation and deliver them specifically to human liver cells in vitro and in vivo [32]. The final subcellular destination of these payloads is the cytoplasm. Unfortunately, encapsulation can be inefficient and inconsistent, probably due to the reduction of membrane fluidity caused by spontaneous formation of multiple 11

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disulfide bonds between S regions. Since 8 out of 14 Cys residues in the S region were found to form excessive disulfide bonds in the BNC during long-term storage, these Cys

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residues were substituted by Ser and Ala to improve the efficiency of electroporation

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[54]. The mutated BNC showed higher stability and more efficient gene delivery

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compared to the wild type. However, because large-scale production of BNC-based nanomedicines would require a massive and impractical electroporation system, we focused our attention on developing other encapsulation methods.

Genetic fusion

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2.4.2.

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For protein delivery only, L protein could be fused with a protein of interest by using recombinant DNA technology. When EGFP (as a model payload) was fused with the C-terminal of L protein, the EGFP-fused BNCs were successfully synthesized as

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~100-nm-sized spherical particles by COS-7 cells [57]. When transfected to various

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cells in vitro, the EGFP-fused BNCs accumulated specifically and efficiently in human hepatic cells. Because these payloads cannot be released from BNCs, their final

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subcellular destinations are the endosomes or endosomal membrane. Furthermore, the fusion with an exogenous protein often affects the productivity of BNCs in cells, which is probably due to the fused protein inhibiting L protein translocation across the ER

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membrane. Moreover, C-terminally fused proteins sometimes locate both inside and outside of the BNC, which would destabilize the nanocarrier’s structure.

2.4.3.

Chemical conjugation

Chemical conjugation has been applied to display payloads on the surface of BNCs. At least four free amino groups on the surface of the BNC can be fluorescently labeled with N-hydroxysuccinimide (NHS) ester-conjugated fluorophores to allow visualization of the nanocarrier [43]. To facilitate delivery of payloads, the BNCs and payloads were conjugated with bi-functional crosslinkers (including NHS ester for amine groups and pyridyldithiol for thiol groups). Following reduction of the payload with dithiothreitol, the sulfhydryl-exposed payload was mixed with pyridyldithiol-activated BNCs to form payload-displaying BNCs [58]. These can be 12

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used for human liver-specific payload delivery in vitro and in vivo; however, they

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Liposome fusion

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2.4.4.

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cannot release payloads into the cytoplasm from endocytic vesicles.

For the efficient cytoplasmic delivery of payloads in an intact form, we developed a new encapsulation method as an alternative to electroporation of BNCs. Kaneda et al. [59] demonstrated that Sendai virus could form a complex with LPs, and

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that the complex (i.e., the virosome) could deliver genes in vitro and in vivo. This result led us to examine complex formation between BNCs and LPs. In 2008, our group

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reported spontaneous formation of a BNC-LP complex under neutral conditions at room temperature [60](see Figs. 2B and 2C). Recently, it was revealed that the fusogenic domain of L protein contributes substantially to BNC-LP complex formation (see

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section 2.3.2.) [49]. We have now successfully performed human liver-specific

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cytoplasmic delivery in vitro and in vivo with a BNC-LP complex containing genes and polystyrene beads (~100-nm in size). Since LPs can accept various payloads (e.g., drugs,

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nucleic acids, proteins, and beads) in their intact forms at higher concentrations, the LP fusion method could further expand the potential of BNC-based nanomedicines. In practical terms, BNC-LP complexes can deliver doxorubicin in vivo [61], siRNA in vitro

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[62], and miRNA in vitro [63] in a human liver-specific manner and subsequently show significant biological functions. These results strongly suggest that the BNC portion of the complex enhances the cellular attachment, internalization, membrane fusion, and endosomal escape of LPs. Moreover, the LP method requires no special equipment, and it has the potential to be upscaled for mass production. The incorporation of LP to BNC allowed us to change the diameter of BNC-LP complex from ~500 nm to ~80 nm by using extruder. Katayama et al. [64] developed a novel cationic polymer for gene delivery that released genes in response to the phosphorylation activity of protein kinase A (PKA) and protein kinase Cα (PKCα). BNC-LP complexes that contained polymer-DNA complexes achieved not only targeted delivery to human liver cells but also controlled gene expression via local phosphorylation activity [65,66]. Since the activity of PKA and PKCα in cells is often enhanced in many types of cancers, this strategy could be 13

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useful for cancer-specific gene therapy. Thus, BNC-LP complexes could be applied to induce microenvironment-dependent gene expression by using intracellular

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Virosome formation

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signal-responsive polymers.

In the process of optimizing formation of the BNC-LP complex, our group discovered that complexes prepared using low pH and high temperatures exhibited a

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unique structure when viewed with a transmission electron microscope [67](see Fig. 4D). Compared with previous versions of the BNC-LP complex (i.e., complexes

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prepared under neutral conditions at room temperature; see Fig. 4C), the morphology had changed dramatically from rugged to smooth spheres, whereas the molecular weight and membrane topology of the L protein remained unchanged. Since the fusogenic

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activity of the pre-S1 region is dependent on low pH [49], and high temperatures

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enhance the fluidity of both membranes, these results indicated that the L proteins of BNCs were disassembled and transferred to LPs with correct membrane topology. In

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addition, the surface structure of the complexes was quite similar to those of the BNC and HBV. In order to discriminate the new version of the BNC-LP complex from the previous version, we designated it as a virosome. Because virosomes possess a higher

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content of phospholipids, the encapsulation of drugs could be carried out by using a conventional remote loading technique, and the diameter of virosomes could be easily changed from ~500 nm to ~80 nm by using extruder. When compared with BNC-LP complexes containing doxorubicin, virosomes containing doxorubicin showed similar anti-tumor effects specific to human liver cells in vivo, but these effects were observed at lower doses and with fewer side effects [67]. In particular, the anti-tumor effect was significantly enhanced, in a synergistic manner, when virosomes were used in combination with radiotherapy.

2.4.6.

Polymer fusion

Polyethyleneimine (PEI) has been widely used for gene delivery because of its high transfection efficiency [68]. The positive charge of PEI contributes to non-specific 14

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cellular attachment and cell entry, as well as proton buffering in late endosomes/lysosomes, and rupture of late endosomes/lysosomes leading to endosomal

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escape (the proton sponge effect) [69]. Since PEI lacks targeting machinery, its in vivo

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use has been limited. However, PEI can be mixed with BNCs, and subsequently with

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DNA, to form BNC-PEI-DNA complexes. These complexes can successfully deliver human liver cell-specific DNA in vitro, which escapes from endocytic vesicles and is ultimately involved gene expression [70]. Thus, BNCs could be used to endow

Retargeting of BNCs for various targets

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2.5.

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polymeric nanocarriers with the ability to target human liver cells.

In addition to utilizing BNCs as human hepatic cell-specific nanocarriers, we have developed retargeting strategies for the treatment of various diseases (Fig. 5).

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Initially, we generated epidermal growth factor (EGF)-displaying BNCs by replacing

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the human hepatocyte-recognition domain of L protein (3–77 aa) with EGF via genetic modification [32]. The EGF-displaying BNC was capable of delivering fluorescent dye

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specifically to EGFR-overexpressing cells in vitro. However, the insertion of exogenous proteins into the pre-S region sometimes reduces the efficiency of membrane translocation of the L protein, and thereby inhibits BNC biosynthesis. In a 2007 study,

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the tandem form of the IgG Fc-binding domain (ZZ domain), derived from Staphylococcus aureus protein A, did not inhibit BNC biosynthesis; on the contrary, facilitating the production of sufficient amounts of modified BNC (ZZ-BNC) [71]. ZZ-BNCs displayed antibodies by tethering the Fc region onto its surface and outwardly deploying the Fv regions. When xenograft mice were injected intracerebroventricularly with ZZ-BNCs displaying anti-EGFR antibodies, the antibodies successfully accumulated in EGFR-expressing glioma in the brain. Furthermore, these ZZ-BNCs successfully delivered a soluble taxol derivative and EGFP, incorporated by electroporation and genetic fusion respectively, to EGFR-expressing cells in vitro [72,73]. The aforementioned antibody-based retargeting strategy is also effective for enhancing the immunogenicity of vaccines in vivo. Dendritic cells (DCs) play a crucial role in the function of the immune system; thus, delivery of DC-specific antigens can 15

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induce efficient immunoreaction against pathogens and cancers [74]. CD11c is a specific marker for DCs. In a previous study, anti-CD11c antibodies were displayed on

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ZZ-BNCs (anti-DC-BNCs) and intravenously injected into mice; results showed that

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anti-DC-BNCs accumulated to >60% of splenic DCs [75]. Subsequently, anti-DC-BNCs

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were formulated into a vaccine via the fusion of LP with the antigen. The vaccine elicited antibody production that was significantly greater than that of the antigen alone, the LP-antigen complex, or the ZZ-BNC-LP-antigen complex. Recently, it was revealed that the ZZ-BNC acts as a scaffold for antibodies in an oriented-immobilization manner,

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with the Fv regions being assembled into closely packed arrays on the surface of the ZZ-BNC [76,77], and it significantly enhanced both the avidity and antigen-binding

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capacity of antibodies. When three α-helices in the Z domain of the ZZ-BNC were rationally modified to recognize oncoprotein HER2 (i.e., an affibody), the affibody-displaying BNCs were capable of HER2-dependent fluorophore delivery in

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vitro. Thus, the affibody-displaying ZZ-BNC is a promising alternative to the ZZ-BNC

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that no longer requires full-sized IgG [78]. Various molecules have also been applied to facilitate retargeting of BNCs.

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One example is an IgG Fc-fused ligand, chlorotoxin (a 36-aa peptide derived from Leiurus quinquestriatus [scorpion] venom), which preferentially binds to matrix metalloprotease 2 (MMP2) on the cell surface and shows cytotoxicity [79]. When

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chlorotoxin was fused with the IgG Fc region, ZZ-BNCs displaying chlorotoxin-fused Fc proteins were able to attach through MNP2 to glioma cells in vitro and then enter into cells via receptor-mediated endocytosis; consequently, cell growth was inhibited [80]. A second example is a lectin, Phaseolus vulgaris agglutinin-L4 isolectin. This binds to β1-6 branching N-acetylglucosamine (GlcNAc), which is abundantly expressed in highly metastatic tumor cells. When ZZ-BNCs were chemically modified with this lectin by way of avidin-biotin complex chemistry, the modified ZZ-BNCs delivered genes into β1-6 GlcNAc-expressing cells in vitro and accumulated at malignant tumors in vivo [81]. Another molecule used for retargeting is a nanobody derived from variable domains of camelid heavy chain antibody. In this example, the 15-aa biotin-acceptor peptide (BAP) was genetically replaced with the 33–159 aa of the L protein. After the enzymatic ligation of biotin, the biotinylated BAP-modified BNC was further modified with biotinylated anti-EGFR nanobodies by using streptavidin. This 16

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nanobody-displaying BNC was found to accumulate in EGFR-expressing cells in vitro [82]. By using a combination of this BAP-modified BNC and enzymatic biotinylation, it

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should be possible to conjugate diverse ligands for the retargeting of BNCs. Taken

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together, these examples confirm that by displaying various bio-recognition molecules

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such as antibodies, affibodies, IgG Fc-fused ligands, lectins, and nanobodies, BNCs can be retargeted to cells and tissues of interest for in vitro and in vivo delivery of payloads. Hence, BNCs could produce therapeutic effects in tissues other than the human liver via the delivery of drugs, nucleic acids, and proteins. However, it should be noted that the

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surface modifications described above would change the physicochemical properties of BNCs (diameter, -potential) and affect the in vitro/in vivo behaviors. We should

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carefully optimize the retargeting molecules not to induce unexpected side effects in vitro and in vivo.

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Modification of BNCs for efficient intracellular delivery

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2.6.

Along with targeted drug and gene delivery, non-specific delivery is considered

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important for some specialized clinical purposes, including ex vivo gene therapy and adoptive immunotherapy. Cell penetrating peptide (CPP) activity has been identified in synthetic octaarginine peptide, HIV-1-derived transactivator protein, and Drosophila

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melanogaster Antennapedia-homeodomain protein, and these have been used as tags for non-specific intracellular drug delivery. Following modification with these CPPs, BNCs were examined for in vitro cellular uptake [83]. Octaarginine-modified BNCs showed the highest cellular uptake efficiency among a wide variety of cells, but they did not exist uniformly in the cytoplasm and accumulated in unidentified subcellular compartments. As described above, where nanocarriers enter cells via endocytosis, endosomal escape is one of the most critical steps towards intracellular drug delivery. Fusion with, or disruption of, the endosomal membrane is essential; escape from endocytic vesicles to the cytoplasm should follow [84]. Although both BNCs and HBV already possess the ability to introduce payloads and viral genomes to human hepatic cells [29], respectively, the potential enhancement of endosomal escape in vitro was also examined by further modifying the affibody-displaying BNC (see section 2.5.) with GALA, a low 17

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pH-dependent fusogenic peptide [85]. In contrast with the affibody-displaying BNC, the GALA-modified BNC preferentially localized to the cytoplasm. This result suggests

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that both targeting and intracellular delivery of BNCs could be improved concurrently

3.

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by conjugation with targeting molecules and low pH-dependent fusogenic peptides.

Virus-inspired nanocarriers

The development of BNC technology has thus far aimed to realize the five

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prerequisite capabilities of an ideal nanocarrier (see section 1.) by biomimicking the infection machinery of HBV. Meanwhile, other researchers have also developed

Virus-like particles

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3.1.

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based on their structures (Fig. 6).

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virus-inspired nanocarriers [86], which can be classified into at least three categories

Virus-like particles (VLPs) are composed of virus-derived structural proteins

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(i.e., envelopes or capsids), which can be synthesized in exogenous cells and subsequently self-assembled into nanoparticles in vitro (Fig. 6A). Numerous types of VLP have so far been developed using recombinant DNA technology. Some retain the

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infection ability derived from their parental virus, but they do not replicate in cells because they lack genetic material. Since VLPs contain virus-specific antigens, they have been utilized in vaccines for humans and animals [87]. Because the social demand for nanomedicines has increased, VLPs have recently been considered as a platform for DDS nanocarriers. For example, human papillomavirus (HPV) major protein L1 has been expressed in insect cells as ~50-nm-sized VLPs [88]. These VLPs were disassembled in dithiothreitol and ethylene glycol tetraacetic acid, and the encapsulation of plasmid DNA was achieved in the presence of CaCl2 and dimethyl sulfoxide. The plasmid DNA-encapsulated VLPs transduced gene expression in various cells in vitro with significantly higher efficiency than conventional liposomal transfection reagents. Similarly, simian virus 40 (SV40)-derived VP1/2/3 proteins have been synthesized in insect cells as ~40-nm-sized VLPs. Each VP protein incorporated plasmid DNA during CaCl2-induced VLP assembly, and the VLPs were applied as gene delivery nanocarriers 18

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in vitro [89-91]. The majority of VLPs showed high transfection efficiency; however, they lacked stringent cell specificity. Therefore, SV40 VP1-derived VLPs were

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chemically modified with human EGF and retargeted to EGFR-expressing cells in vitro

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[92]. The results of these studies suggest that VLPs have the potential to act as active

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targeting-based nanocarriers. However, it remains unclear how VLPs enter cells, move intracellularly, and release payloads. To enable the use of VLPs in vivo, it will be necessary to lower their immunogenicity and endow them with the ability for stealth

Virosomes

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3.2.

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activity.

A virosome is an artificial hollow nanocapsule composed of viral membrane-associated proteins and an exogenous LP; these structures were first

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generated by mixing influenza virus subunits (hemagglutinin and neuraminidase) and

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LPs [93]. Virosomes are approximately 150 nm in diameter and possess morphology similar to that of the original influenza virion, with hemagglutinins and neuraminidases

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displayed on their surface in the proper direction. Since virosomes are prepared without hazardous viral genomes, they are considered as safe biomaterials (Fig. 6B). Thus, virosomes originating from influenza virus have already been commercialized as an

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influenza vaccine [94]. In one study, following the adsorption of hepatitis A virus antigens on the surface of influenza-derived virosomes, the virosomes produced an immune response against hepatitis A virus in mice that was significantly higher than that provided by a conventional vaccine formulation [95]. Kaneda et al. [59] have also prepared ~500-nm virosomes with Sendai virus, LPs containing plasmid DNA, and erythrocyte ghosts containing nuclear proteins,. They succeeded in inducing gene expression in the liver of rats by injecting virosomes through the portal vein, which indicated that they could be used as DDS nanocarriers. In another study, influenza-derived virosomes (80–200 nm in diameter) were modified with PEGylated antibodies against oncoprotein HER2 and then loaded with doxorubicin using a remote loading method; these virosomes were able to deliver doxorubicin specifically to tumors and inhibit tumor growth following intravenous injection [96]. Current evidence therefore indicates that virosomes can be loaded with various payloads, such as drugs, 19

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nucleic acids, proteins, simultaneously; that they can fuse with the plasma membrane owing to pH-independent fusogenic activity; and ultimately release their payloads into

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the cytoplasm directly [97]. Through a process of surface modification with targeting

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molecules and PEG, virosomes can be endowed with the capability for active targeting

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and stealth activity. However, several types of membrane-associated proteins are required for their formation (e.g., hemagglutinin and neuraminidase for influenza virus, and F and M proteins for Sendai virus), and these can unexpectedly produce larger and increasingly immunogenic virosomes. Where virosomes are applied clinically, their

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viral components should be trimmed, conserving only those domains essential for

3.3.

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virosomal functions, and the ratio of viral components to LPs should be optimized.

Virus-mimicking nanocarriers

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As well as utilizing actual viral components, several attempts have been made

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to generate complete artificial nanocarriers inspired by the functional domains of viral components (Fig. 6C). For example, both human ferritin light chain and heat shock

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protein (Hsp) 16.5 from a hyperthermophilic archaeon were able to form ~20-nm-sized nanocages and incorporate therapeutic molecules into the internal hollow space [98,99]. Following fusion with the pre-S1 peptide (10–36 aa) of HBV, these nanocages

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accumulated in human hepatic cells in vitro [100,101]. Titanium dioxide nanoparticles (~100 nm in diameter) have also been modified with the same pre-S1 peptides, and they too accumulated specifically to human hepatic cells in vitro [102]. Given ultrasound irradiation, titanium dioxide nanoparticles can generate radicals; thus, intratumorally injected nanoparticles showed cytotoxic effects in a xenograft mice following ultrasound irradiation [103]. Since the pre-S1 peptide (myristoylated 2–48 aa) was shown to interact with NTCP [38] (see section 2.3.1.), LPs were recently functionalized with the pre-S1 peptide for targeting liver cells. After loading with silybin, the LPs were administered intravenously to mice suffering from carbon tetrachloride-induced acute liver damage; LPs were found to accumulate preferentially in the mouse liver, and they showed therapeutic effects on liver damage [104]. The aforementioned nanocarriers are prepared by simply conjugating artificial nanocapsules with viral targeting domains (mainly from the HBV pre-S1 region). They have several advantages, including the 20

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acceptance of various types of payload, the ability to repel the adsorption of opsonins by PEG modification, and reduced immunogenicity compared with VLPs or virosomes.

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However, they are fundamentally similar to the conventional active targeting-based

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nanocarriers (e.g., transferrin-conjugated LPs [14] and folate-conjugated LPs [15]).

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Therefore, in future research, new virus-mimicking nanocarriers should be designed rationally to possess the capabilities of an ideal nanocarrier (see section 1.) by merging artificial nanocapsules with viral functional domains and/or their artificial orthologs. Harashima’s group have extensively analyzed the rate-limiting cellular events

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for gene delivery with LPs [105] and optimized the components of LPs for each event [106]. For example, stealth activity was provided by PEG modification, targeting

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activity by aptamer modification [107], enhanced cellular uptake activity by octaarginine peptide and fusogenic lipids [108], and efficient endosomal escape activity by low pH-dependent fusogenic GALA peptide [109]. Moreover, they have established

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a programmed-packaging protocol for realizing these activities hierarchically in LPs,

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and also obtained a multi-functional envelope-type nano device (MEND) that significantly improves transfection efficiency (to levels comparable to adenovirus) in

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vitro [108] and achieves efficient gene silencing in pulmonary endothelial cells via systemic siRNA administration [109]. Thus, MEND is considered a state-of-art

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virus-mimicking nanocarrier.

4. Conclusion

The following five functions, inherent in viruses, should be considered the prerequisite capabilities of forthcoming nanocarriers: evasion of the host immune system, active targeting, entering cells, escaping from endosomes/lysosomes to the cytoplasm, and releasing payloads. BNCs are potentially capable of all of the above. As summarized in Table 2, BNC technology facilitates the targeted delivery of various types of payload in vitro and in vivo, which demonstrates that BNCs are one of the most promising virus-inspired nanocarriers. Indeed, the HBV envelope M protein particle synthesized in yeast, of which the essential structure is the same as the BNC except pre-S1 region, has been confirmed as a non-toxic and safe biomaterial and used as an immunogen in clinical trials [24]. Since the expression system and purification protocol 21

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of BNC are essentially based on those of M particle, it is possible to produce BNC under the same quality control of M particle, and it is therefore postulated that BNC is

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also non-toxic and safe material for human. However, the following issues of BNC

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should be addressed before moving forward to clinical trials. First, the pharmacokinetics

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and pharmacodynamics of BNC-based medicines should be optimized for maximizing the therapeutic efficacy. Second, the immunogenicity should be lowered for the repetitive administration. Third, the production cost should be lowered by simplifying CMC (chemistry, manufacturing and control), because biological materials (i.e., BNC)

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are often more expensive than chemical materials. Considering the qualities of virus-mimicking nanocarriers (especially MEND), BNCs could be improved by

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eliminating immunogenicity with proper trimming that conserves functional domains. Ideally, to reconstitute viral functions, the essential functional domains of BNCs should be prepared using chemically defined synthetic materials and deployed in an optimized

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spatial arrangement on the surface of artificial nanocarriers (e.g., LPs). These strategies

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might lower the immunogenicity and production cost of virus-inspired nanocarriers including BNC. Additionally, the potential of BNC technology could be further

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expanded if the nuclear translocation of genes was enhanced by motor proteins (e.g., dynein) and nuclear localization signals, and if direct cytoplasmic drug delivery and organelle-specific drug delivery became available [110]. In the future, an improved

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understanding of the behaviors of viruses will enable the development of new virus-inspired nanocarriers that can overcome the problems of conventional synthetic nanocarriers and may represent effective platforms for drug delivery.

Acknowledgments

This work was supported in part by The Naito Foundation (to S.K.), The Okawa Foundation (to S.K.), KAKENHI (Grant-in-Aid for Scientific Research [A], 25242043, to S.K.; Grant-in-Aid for JSPS Fellows, 25003835, to M.S.), and a Health Labor Sciences Research Grant from the Ministry of Health Labor and Welfare (to S.K.).

References 22

ACCEPTED MANUSCRIPT

[1]

Y. Zhang, H.F. Chan, K.W. Leong, Advanced materials and processing for drug

IP

T

delivery: the past and the future., Adv. Drug Deliv. Rev. 65 (2013) 104–20. doi:10.1016/j.addr.2012.10.003. D. Peer, J.M. Karp, S. Hong, O.C. Farokhzad, R. Margalit, R. Langer, Nanocarriers as an emerging platform for cancer therapy., Nat. Nanotechnol. 2 (2007) 751–760. doi:10.1038/nnano.2007.387.

[3]

L. Brannon-Peppas, J.O. Blanchette, Nanoparticle and targeted systems for cancer therapy, Adv. Drug Deliv. Rev. 64 (2012) 206–212. doi:10.1016/j.addr.2012.09.033.

[4]

A. Gabizon, R. Catane, B. Uziely, B. Kaufman, T. Safra, R. Cohen, et al., Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene-glycol coated liposomes, Cancer Res. 54 (1994) 987–992.

[5]

Y. Barenholz, Doxil - The first FDA-approved nano-drug: Lessons learned, J. Control. Release. 160 (2012) 117–134. doi:10.1016/j.jconrel.2012.03.020.

[6]

H. Cabral, K. Kataoka, Progress of drug-loaded polymeric micelles into clinical studies, J. Control. Release. 190 (2014) 465–476. doi:10.1016/j.jconrel.2014.06.042.

[7]

V.P. Torchilin, Recent advances with liposomes as pharmaceutical carriers., Nat. Rev. Drug Discov. 4 (2005) 145–160. doi:10.1038/nrd1632.

[8]

K. a Whitehead, R. Langer, D.G. Anderson, Knocking down barriers: advances in siRNA delivery., Nat. Rev. Drug Discov. 8 (2009) 129–38. doi:10.1038/nrd2742.

[9]

Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cnacer chemotherapy: mechanism of tumoritropic accumulatio of proteins and the antitumor agents Smancs, Cancer Res. 46 (1986) 6387– 6392. http://cancerres.aacrjournals.org/content/46/12_Part_1/6387.short (accessed January 22, 2015).

AC

CE P

TE

D

MA

NU

SC R

[2]

23

ACCEPTED MANUSCRIPT

IP

T

[10] H. Maeda, The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting., Adv. Enzyme Regul. 41 (2001) 189–207. http://www.ncbi.nlm.nih.gov/pubmed/11384745.

NU

SC R

[11] H. Maeda, T. Sawa, T. Konno, Mechanism of tumor-targeted delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANCS, J. Control. Release. 74 (2001) 47–61. doi:10.1016/S0168-3659(01)00309-1.

MA

[12] H. Maeda, Toward a full understanding of the EPR effect in primary and metastatic tumors as well as issues related to its heterogeneity, Adv. Drug Deliv. Rev. (2015) 2–5. doi:10.1016/j.addr.2015.01.002.

D

[13] R.K. Jain, Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumors, Cancer Res. 50 (1990).

CE P

TE

[14] Z.M. Qian, H. Li, H. Sun, K. Ho, Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway., Pharmacol. Rev. 54 (2002) 561–587. doi:10.1124/pr.54.4.561.

AC

[15] L. Teng, J. Xie, L. Teng, R.J. Lee, Clinical translation of folate receptor-targeted therapeutics, Expert Opin. Drug Deliv. 9 (2012) 901–908. doi:10.1517/17425247.2012.694863. [16] J.D. Heidel, Z. Yu, J.Y.-C. Liu, S.M. Rele, Y. Liang, R.K. Zeidan, et al., Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA., Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 5715–21. doi:10.1073/pnas.0701458104. [17] M.E. Davis, J.E. Zuckerman, C.H.J. Choi, D. Seligson, A. Tolcher, C. a Alabi, et al., Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles., Nature. 464 (2010) 1067–1070. doi:10.1038/nature08956. [18] O. Germershaus, T. Merdan, U. Bakowsky, M. Behe, T. Kissel, Trastuzumab-polyethylenimine-polyethylene glycol conjugates for targeting 24

ACCEPTED MANUSCRIPT

T

Her2-expressing tumors., Bioconjug. Chem. 17 (2006) 1190–9. doi:10.1021/bc0601119.

SC R

IP

[19] R. Kanasty, J.R. Dorkin, A. Vegas, D. Anderson, Delivery materials for siRNA therapeutics., Nat. Mater. 12 (2013) 967–77. doi:10.1038/nmat3765. [20] J. Schneider-Schaulies, Cellular receptors for viruses: Links to tropism and pathogenesis, J. Gen. Virol. 81 (2000) 1413–1429.

NU

[21] P. Tiollais, C. Pourcel, A. Dejean, The hepatitis B virus, Nature. 317 (1985) 489–495. doi:10.1038/317489a0.

MA

[22] P. Valenzuela, A. Medina, W.J. Rutter, G. Ammerer, B.D. Hall, Synthesis and assembly of hepatitis B virus surface antigen particles in yeast., Nature. 298 (1982) 347–350. doi:10.1038/298347a0.

D

[23] Y. Itoh, Y. Fujisawa, Synthesis in yeast of hepatitis B virus surface antigen

TE

modified P31 particles by gene modification., Biochem. Biophys. Res. Commun. 141 (1986) 942–948. doi:10.1016/S0006-291X(86)80134-6.

AC

CE P

[24] S. Kuroda, Y. Fujisawa, S. Iino, Y. Akahane, H. Suzuki, Induction of protection level of anti-pre-S2 antibodies in humans immunized with a novel hepatitis B vaccine consisting of M (pre-S2 + S) protein particles (a third generation vaccine), Vaccine. 9 (1991) 163-169. [25] S. Kuroda, S. Otaka, T. Miyazaki, M. Nakao, Y. Fujisawa, Hepatitis B virus envelope L protein particles: Synthesis and assembly in Saccharomyces cerevisiae, purification and characterization, J. Biol. Chem. 267 (1992) 1953–1961. http://www.ncbi.nlm.nih.gov/pubmed/18434744. [26] J. Jung, M. Iijima, N. Yoshimoto, M. Sasaki, T. Niimi, K. Tatematsu, et al., Efficient and rapid purification of drug- and gene-carrying bio-nanocapsules, hepatitis B virus surface antigen L particles, from Saccharomyces cerevisiae., Protein Expr. Purif. 78 (2011) 149–55. doi:10.1016/j.pep.2011.04.008. [27] T. Yamada, H. Iwabuki, T. Kanno, H. Tanaka, T. Kawai, H. Fukuda, et al., Physicochemical and immunological characterization of hepatitis B virus

25

ACCEPTED MANUSCRIPT

T

envelope particles exclusively consisting of the entire L (pre-S1+pre-S2+S) protein, Vaccine. 19 (2001) 3154–3163. doi:10.1016/S0264-410X(01)00017-2.

SC R

IP

[28] Y.E. Cossart, A.M. Field, Virus-like particles in serum of patients with Australia-antigen-associated hepatitis., Lancet. 1 (1970) 848. doi:10.1016/S0140-6736(70)92460-8. [29] K. Watashi, S. Urban, W. Li, T. Wakita, NTCP and beyond: opening the door to

NU

unveil hepatitis B virus entry., Int. J. Mol. Sci. 15 (2014) 2892–905. doi:10.3390/ijms15022892.

MA

[30] A.R. Neurath, S.B. Kent, N. Strick, K. Parker, Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus., Cell. 46 (1986) 429–436. doi:0092-8674(86)90663-X [pii].

D

[31] A.R. Neurath, B. Seto, N. Strick, Antibodies to synthetic peptides from the preS1 region of the hepatitis B virus (HBV) envelope (env) protein are

CE P

TE

virus-neutralizing and protective., Vaccine. 7 (1989) 234–236. doi:10.1016/0264-410X(89)90235-1.

AC

[32] T. Yamada, Y. Iwasaki, H. Tada, H. Iwabuki, M.K.L. Chuah, T. VandenDriessche, et al., Nanoparticles for the delivery of genes and drugs to human hepatocytes., Nat. Biotechnol. 21 (2003) 885–890. doi:10.1038/nbt843. [33] Y. Iwasaki, M. Ueda, T. Yamada, a Kondo, M. Seno, K. Tanizawa, et al., Gene therapy of liver tumors with human liver-specific nanoparticles., Cancer Gene Ther. 14 (2007) 74–81. doi:10.1038/sj.cgt.7701031. [34] Y. Matsuura, H. Yagi, S. Matsuda, O. Itano, K. Aiura, S. Kuroda, et al., Human liver-specific nanocarrier in a novel mouse xenograft model bearing noncancerous human liver tissue., Eur. Surg. Res. 46 (2011) 65–72. doi:10.1159/000322491. [35] A. Schulze, P. Gripon, S. Urban, Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans., Hepatology. 46 (2007) 1759–1768. doi:10.1002/hep.21896.

26

ACCEPTED MANUSCRIPT

T

[36] J. Salisse, C. Sureau, A function essential to viral entry underlies the hepatitis B virus ―a‖ determinant., J. Virol. 83 (2009) 9321–8. doi:10.1128/JVI.00678-09.

SC R

IP

[37] M. Bernfield, M. Götte, P.W. Park, O. Reizes, M.L. Fitzgerald, J. Lincecum, et al., Functions of cell surface heparan sulfate proteoglycans., Annu. Rev. Biochem. 68 (1999) 729–777. doi:10.1146/annurev.biochem.68.1.729. [38] H. Yan, G. Zhong, G. Xu, W. He, Z. Jing, Z. Gao, et al., Sodium taurocholate

NU

cotransporting polypeptide is a functional receptor for human hepatitis B and D virus., Elife. 1 (2012) e00049. doi:10.7554/eLife.00049.

MA

[39] M. Iwamoto, K. Watashi, S. Tsukuda, H.H. Aly, M. Fukasawa, A. Fujimoto, et al., Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP, Biochem. Biophys. Res. Commun. 443 (2014) 808–813. doi:10.1016/j.bbrc.2013.12.052.

D

[40] H. Yan, B. Peng, W. He, G. Zhong, Y. Qi, B. Ren, et al., Molecular determinants

CE P

TE

of hepatitis B and D virus entry restriction in mouse sodium taurocholate cotransporting polypeptide., J. Virol. 87 (2013) 7977–91. doi:10.1128/JVI.03540-12.

AC

[41] H. Li, Q. Zhuang, Y. Wang, T. Zhang, J. Zhao, Y. Zhang, et al., HBV life cycle is restricted in mouse hepatocytes expressing human NTCP., Cell. Mol. Immunol. 11 (2014) 175–83. doi:10.1038/cmi.2013.66. [42] A. Meier, S. Mehrle, T.S. Weiss, W. Mier, S. Urban, Myristoylated PreS1-domain of the hepatitis B virus L-protein mediates specific binding to differentiated hepatocytes., Hepatology. 58 (2013) 31–42. doi:10.1002/hep.26181. [43] M. Yamada, A. Oeda, J. Jung, M. Iijima, N. Yoshimoto, T. Niimi, et al., Hepatitis B virus envelope L protein-derived bio-nanocapsules: mechanisms of cellular attachment and entry into human hepatic cells., J. Control. Release. 160 (2012) 322–9. doi:10.1016/j.jconrel.2011.11.004. [44] T. Kasuya, S. Nomura, T. Matsuzaki, J. Jung, T. Yamada, K. Tatematsu, et al., Expression of squamous cell carcinoma antigen-1 in liver enhances the uptake of

27

ACCEPTED MANUSCRIPT

T

hepatitis B virus envelope-derived bio-nanocapsules in transgenic rats., FEBS J. 275 (2008) 5714–24. doi:10.1111/j.1742-4658.2008.06698.x.

SC R

IP

[45] S.C. Harrison, Viral membrane fusion., Nat. Struct. Mol. Biol. 15 (2008) 690–8. doi:10.1038/nsmb.1456. [46] T.F. Baumert, L. Meredith, Y. Ni, D.J. Felmlee, J. a. McKeating, S. Urban, Entry of hepatitis B and C viruses - Recent progress and future impact, Curr. Opin. Virol. 4 (2014) 58–65. doi:10.1016/j.coviro.2013.12.002.

MA

NU

[47] S. Oess, E. Hildt, Novel cell permeable motif derived from the PreS2-domain of hepatitis-B virus surface antigens., Gene Ther. 7 (2000) 750–758. doi:10.1038/sj.gt.3301154.

TE

D

[48] I. Rodriguez-Crespo, J. Gomez-Gutierrez, M. Nieto, D.L. Peterson, F. Gavilanes, Prediction of a putative fusion peptide in the S protein of hepatitis B virus, J. Gen. Virol. 75 (1994) 637–639. doi:10.1099/0022-1317-75-3-637.

AC

CE P

[49] M. Somiya, Y. Sasaki, T. Matsuzaki, Q. Liu, M. Iijima, N. Yoshimoto, et al., Intracellular traficking of bio-nanocapsule-liposome complex: Identification of fusogenic activity in the pre-S1 region of hepatitis B virus surface antigen L protein, J. Control. Release. 212 (2015) 10-18. doi:10.1016/j.jconrel.2015.06.012. [50] B. Krone, a Lenz, K.H. Heermann, M. Seifer, X.Y. Lu, W.H. Gerlich, Interaction between hepatitis B surface proteins and monomeric human serum albumin., Hepatology. 11 (1990) 1050–6. http://www.ncbi.nlm.nih.gov/pubmed/2163967. [51] K. Ishihara, J. a Waters, M. Pignatelli, H.C. Thomas, Characterisation of the polymerised and monomeric human serum albumin binding sites on hepatitis B surface antigen., J. Med. Virol. 21 (1987) 89–95. http://www.ncbi.nlm.nih.gov/pubmed/3794674. [52] Y. Itoh, S. Kuroda, T. Miyazaki, S. Otaka, Y. Fujisawa, Identification of polymerized-albumin receptor domain in the pre-S2 region of hepatitis B virus surface antigen M protein., J. Biotechnol. 23 (1992) 71–82. doi:10.1016/0168-1656(92)90100-N.

28

ACCEPTED MANUSCRIPT

IP

T

[53] E.T. Dams, P. Laverman, W.J. Oyen, G. Storm, G.L. Scherphof, J.W. van Der Meer, et al., Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes., J. Pharmacol. Exp. Ther. 292 (2000) 1071–1079.

NU

SC R

[54] T. Nagaoka, T. Fukuda, S. Yoshida, H. Nishimura, D. Yu, S. Kuroda, et al., Characterization of bio-nanocapsule as a transfer vector targeting human hepatocyte carcinoma by disulfide linkage modification., J. Control. Release. 118 (2007) 348–56. doi:10.1016/j.jconrel.2006.12.020.

MA

[55] M. Kobayashi, T. Asano, M. Utsunomiya, Y. Itoh, Y. Fujisawa, O. Nishimura, et al., Recombinant hepatitis B virus surface antigen carrying the pre-S2 region derived from yeast: purification and characterization, J. Biotechnol. 8 (1988) 1–21. doi:10.1016/0168-1656(88)90064-8.

TE

D

[56] T. Yamada, J. Jung, M. Seno, A. Kondo, M. Ueda, K. Tanizawa, et al., Electroporation and use of hepatitis B virus envelope L proteins as bionanocapsules, Cold Spring Harb. Protoc. (2012). doi:10.1101/pdb.prot069534.

CE P

[57] D. Yu, C. Amano, T. Fukuda, T. Yamada, S. Kuroda, K. Tanizawa, et al., The specific delivery of proteins to human liver cells by engineered bio-nanocapsules, FEBS J. 272 (2005) 3651–3660. doi:10.1111/j.1742-4658.2005.04790.x.

AC

[58] T. Miyata, S. Tafuku, T. Harakuni, M. Tadano, N. Yoshimoto, M. Iijima, et al., A bio-nanocapsule containing envelope protein domain III of Japanese encephalitis virus protects mice against lethal Japanese encephalitis virus infection, Microbiol. Immunol. 57 (2013) 470–477. doi:10.1111/1348-0421.12055. [59] Y. Kaneda, K. Iwai, T. Uchida, Increased expression of DNA cointroduced with nuclear protein in adult rat liver, Science (80-. ). 243 (1989) 375–378. doi:10.1126/science.2911748. [60] J. Jung, T. Matsuzaki, K. Tatematsu, T. Okajima, K. Tanizawa, S. Kuroda, Bio-nanocapsule conjugated with liposomes for in vivo pinpoint delivery of various materials, J. Control. Release. 126 (2008) 255–264. doi:10.1016/j.jconrel.2007.12.002.

29

ACCEPTED MANUSCRIPT

IP

T

[61] T. Kasuya, J. Jung, R. Kinoshita, Y. Goh, T. Matsuzaki, M. Iijima, et al., Bio-Nanocapsule-Liposome Conjugates for In Vivo Pinpoint Drug and Gene Delivery, Methods Enzymol. 464 (2009) 147–166. doi:10.1016/S0076-6879(09)64008-8.

NU

SC R

[62] M. Somiya, K. Yamaguchi, Q. Liu, T. Niimi, A. Maturana Daniel, M. Iijima, et al., One-step scalable preparation method for non-cationic liposomes with high siRNA content, Int. J. Pharm. 490 (2015) 316-323. doi:10.1016/j.ijpharm.2015.05.072.

MA

[63] M. Ohno, M. Otsuka, T. Kishikawa, C. Shibata, Specific delivery of microRNA93 into HBV-replicating hepatocytes downregulates protein expression of liver cancer susceptible gene MICA, Oncotarget. 5 (2014) 5581–5590. www.impactjournals.com/oncotarget/.

TE

D

[64] Y. Katayama, K. Fujii, E. Ito, S. Sakakihara, T. Sonoda, M. Murata, et al., Intracellular signal-responsive artificial gene regulation for novel gene delivery, Biomacromolecules. 3 (2002) 905–909. doi:10.1021/bm025532h.

AC

CE P

[65] J. Oishi, J. Jung, A. Tsuchiya, R. Toita, J.H. Kang, T. Mori, et al., A gene-delivery system specific for hepatoma cells and an intracellular kinase signal based on human liver-specific bionanocapsules and signal-responsive artificial polymer, Int. J. Pharm. 396 (2010) 174–178. doi:10.1016/j.ijpharm.2010.06.012. [66] J.H. Kang, J. Oishi, J.H. Kim, M. Ijuin, R. Toita, B. Jun, et al., Hepatoma-targeted gene delivery using a tumor cell-specific gene regulation system combined with a human liver cell-specific bionanocapsule, Nanomedicine Nanotechnology, Biol. Med. 6 (2010) 583–589. doi:10.1016/j.nano.2010.01.007. [67] Q. Liu, J. Jung, M. Somiya, M. Iijima, N. Yoshimoto, N. Tomoaki, et al., Virosomes of hepatitis B virus envelope L proteins containing doxorubicin: synergistic enhancement of human liver-specific anti-tumor growth activity by radiotherapy, Int. J. Nanomedicine. 10 (2015) 4159-4172. doi: 10.2147/IJN.S84295. [68] O. Boussif, F. Lezoualc’h, M.A. Zanta, M.D. Mergny, D. Scherman, B. Demeneix, et al., A versatile vector for gene and oligonucleotide transfer into 30

ACCEPTED MANUSCRIPT

IP

T

cells in culture and in vivo: polyethylenimine., Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 7297–7301. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=41326&tool=pmcentr ez&rendertype=abstract.

SC R

[69] A. Akinc, M. Thomas, A.M. Klibanov, R. Langer, Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis, J. Gene Med. 7 (2005) 657–663. doi:10.1002/jgm.696.

MA

NU

[70] M. Somiya, N. Yoshimoto, M. Iijima, T. Niimi, T. Dewa, J. Jung, et al., Targeting of polyplex to human hepatic cells by bio-nanocapsules, hepatitis B virus surface antigen L protein particles., Bioorg. Med. Chem. 20 (2012) 3873–9. doi:10.1016/j.bmc.2012.04.031.

TE

D

[71] Y. Tsutsui, K. Tomizawa, M. Nagita, H. Michiue, T.I. Nishiki, I. Ohmori, et al., Development of bionanocapsules targeting brain tumors, J. Control. Release. 122 (2007) 159–164. doi:10.1016/j.jconrel.2007.06.019.

CE P

[72] H. Hamada, K. Shimoda, M. Seno, Chemo-enzymatic Synthesis of Propionyl-ester-linked Taxol-monosaccharide Conjugate and its Drug Delivery System Using Hybrid-Bio-nanocapsules Targeting Brain Glioma Cells, Clin. Med. Insights Women’s Heal. 6 (2013) 71. doi:10.4137/CMWH.S8213.

AC

[73] N. Kurata, T. Shishido, M. Muraoka, T. Tanaka, C. Ogino, H. Fukuda, et al., Specific protein delivery to target cells by antibody-displaying bionanocapsules, J. Biochem. 144 (2008) 701–707. doi:10.1093/jb/mvn131. [74] P.J. Tacken, I.J.M. de Vries, R. Torensma, C.G. Figdor, Dendritic-cell immunotherapy: from ex vivo loading to in vivo targeting., Nat. Rev. Immunol. 7 (2007) 790–802. doi:10.1038/nri2173. [75] H. Matsuo, N. Yoshimoto, M. Iijima, T. Niimi, J. Jung, S.Y. Jeong, et al., Engineered hepatitis B virus surface antigen L protein particles for in vivo active targeting of splenic dendritic cells, Int. J. Nanomedicine. 7 (2012) 3341–3350. doi:10.2147/IJN.S32813.

31

ACCEPTED MANUSCRIPT

IP

T

[76] M. Iijima, M. Somiya, N. Yoshimoto, T. Niimi, S. Kuroda, Nano-visualization of oriented-immobilized IgGs on immunosensors by high-speed atomic force microscopy., Sci. Rep. 2 (2012) 790. doi:10.1038/srep00790.

SC R

[77] M. Iijima, H. Kadoya, S. Hatahira, S. Hiramatsu, G. Jung, A. Martin, et al., Nanocapsules incorporating IgG Fc-binding domain derived from Staphylococcus aureus protein A for displaying IgGs on immunosensor chips., Biomaterials. 32 (2011) 1455–64. doi:10.1016/j.biomaterials.2010.10.057.

MA

NU

[78] T. Shishido, H. Mieda, S.Y. Hwang, Y. Nishimura, T. Tanaka, C. Ogino, et al., Affibody-displaying bionanocapsules for specific drug delivery to HER2-expressing cancer cells., Bioorg. Med. Chem. Lett. 20 (2010) 5726–31. doi:10.1016/j.bmcl.2010.08.011.

TE

D

[79] J. Deshane, C.C. Garner, H. Sontheimer, Chlorotoxin inhibits glioma cell invasion via matrix metalloproteinase-2, J. Biol. Chem. 278 (2003) 4135–4144. doi:10.1074/jbc.M205662200.

CE P

[80] T. Kasai, K. Nakamura, A. Vaidyanath, L. Chen, S. Sekhar, S. El-Ghlban, et al., Chlorotoxin Fused to IgG-Fc Inhibits Glioblastoma Cell Motility via Receptor-Mediated Endocytosis., J. Drug Deliv. 2012 (2012) 975763. doi:10.1155/2012/975763.

AC

[81] T. Kasuya, J. Jung, H. Kadoya, T. Matsuzaki, K. Tatematsu, T. Okajima, et al., In vivo delivery of bionanocapsules displaying Phaseolus vulgaris agglutinin-L4 isolectin to malignant tumors overexpressing N-acetylglucosaminyltransferase V., Hum. Gene Ther. 19 (2008) 887–95. doi:10.1089/hum.2008.037. [82] T. Shishido, Y. Azumi, T. Nakanishi, M. Umetsu, T. Tanaka, C. Ogino, et al., Biotinylated bionanocapsules for displaying diverse ligands toward cell-specific delivery, J. Biochem. 146 (2009) 867–874. doi:10.1093/jb/mvp134. [83] T. Shishido, D. Yonezawa, K. Iwata, T. Tanaka, C. Ogino, H. Fukuda, et al., Construction of arginine-rich peptide displaying bionanocapsules., Bioorg. Med. Chem. Lett. 19 (2009) 1473–6. doi:10.1016/j.bmcl.2009.01.049.

32

ACCEPTED MANUSCRIPT

IP

T

[84] I.A. Khalil, K. Kogure, H. Akita, H. Harashima, Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery., Pharmacol. Rev. 58 (2006) 32–45. doi:10.1124/pr.58.1.8.32.

SC R

[85] Y. Nishimura, K. Takeda, R. Ezawa, J. Ishii, C. Ogino, A. Kondo, A display of pH-sensitive fusogenic GALA peptide facilitates endosomal escape from a Bio-nanocapsule via an endocytic uptake pathway., J. Nanobiotechnology. 12 (2014) 11. doi:10.1186/1477-3155-12-11.

NU

[86] Y. Seow, M.J. Wood, Biological gene delivery vehicles: beyond viral vectors., Mol. Ther. 17 (2009) 767–77. doi:10.1038/mt.2009.41.

MA

[87] C. Ludwig, R. Wagner, Virus-like particles-universal molecular toolboxes, Curr. Opin. Biotechnol. 18 (2007) 537–545. doi:10.1016/j.copbio.2007.10.013.

TE

D

[88] A. Touze, P. Coursaget, In vitro gene transfer using human papillomavirus-like particles, Nucleic Acids Res. 26 (1998) 1317–1323. doi:10.1093/nar/26.5.1317.

CE P

[89] S.N. Kanesashi, K.I. Ishizu, M.A. Kawano, S.I. Han, S. Tomita, H. Watanabe, et al., Simian virus 40 VP1 capsid protein forms polymorphic assemblies in vitro, J. Gen. Virol. 84 (2003) 1899–1905. doi:10.1099/vir.0.19067-0.

AC

[90] T. Enomoto, I. Kukimoto, M.A. Kawano, Y. Yamaguchi, A.J. Berk, H. Handa, In vitro reconstitution of SV40 particles that are composed of VP1/2/3 capsid proteins and nucleosomal DNA and direct efficient gene transfer, Virology. 420 (2011) 1–9. doi:10.1016/j.virol.2011.08.014. [91] T. Inoue, M.A. Kawano, R.U. Takahashi, H. Tsukamoto, T. Enomoto, T. Imai, et al., Engineering of SV40-based nano-capsules for delivery of heterologous proteins as fusions with the minor capsid proteins VP2/3, J. Biotechnol. 134 (2008) 181–192. doi:10.1016/j.jbiotec.2007.12.006. [92] Y. Kitai, H. Fukuda, T. Enomoto, Y. Asakawa, T. Suzuki, S. Inouye, et al., Cell selective targeting of a simian virus 40 virus-like particle conjugated to epidermal growth factor, J. Biotechnol. 155 (2011) 251–256. doi:10.1016/j.jbiotec.2011.06.030.

33

ACCEPTED MANUSCRIPT

IP

T

[93] J.D. Almeida, D.C. Edwards, C.M. Brand, T.D. Heath, Formation of virosomes from influenza subunits and liposomes, Lancet. 306 (1975) 899–901. http://www.sciencedirect.com/science/article/pii/S0140673675921303 (accessed January 6, 2012).

SC R

[94] D. Felnerova, J.F. Viret, R. Glück, C. Moser, Liposomes and virosomes as delivery systems for antigens, nucleic acids and drugs, Curr. Opin. Biotechnol. 15 (2004) 518–529. doi:10.1016/j.copbio.2004.10.005.

MA

NU

[95] R. Glück, R. Mischler, S. Brantschen, M. Just, B. Althaus, S.J. Cryz, Immunopotentiating reconstituted influenza virus virosome vaccine delivery system for immunization against hepatitis A, J. Clin. Invest. 90 (1992) 2491–2495. doi:10.1172/JCI116141.

TE

D

[96] E. Waelti, N. Wegmann, R. Schwaninger, A. Wetterwald, C. Wingenfeld, B. Rothen-Rutishauser, et al., Targeting HER-2/neu with antirat neu virosomes for cancer therapy, Cancer Res. 62 (2002) 437–444.

CE P

[97] Y. Kaneda, Virosome: A novel vector to enable multi-modal strategies for cancer therapy, Adv. Drug Deliv. Rev. 64 (2012) 730–738. doi:10.1016/j.addr.2011.03.007.

AC

[98] R. Toita, M. Murata, K. Abe, S. Narahara, J.S. Piao, J.H. Kang, et al., Biological evaluation of protein nanocapsules containing doxorubicin, Int. J. Nanomedicine. 8 (2013) 1989–1999. doi:10.2147/IJN.S40239. [99] Z. Zhen, W. Tang, H. Chen, X. Lin, T. Todd, G. Wang, et al., RGD-modified apoferritin nanoparticles for efficient drug delivery to tumors, ACS Nano. 7 (2013) 4830–4837. doi:10.1021/nn305791q. [100] M. Murata, S. Narahara, K. Umezaki, R. Toita, S. Tabata, J.S. Piao, et al., Liver cell specific targeting by the preS1 domain of hepatitis B virus surface antigen displayed on protein nanocages., Int. J. Nanomedicine. 7 (2012) 4353–62. doi:10.2147/IJN.S31365. [101] M. Murata, J.S. Piao, S. Narahara, T. Kawano, N. Hamano, J.-H. Kang, et al., Expression and characterization of myristoylated preS1-conjugated nanocages for

34

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T

targeted cell delivery., Protein Expr. Purif. (2014). doi:10.1016/j.pep.2014.12.001.

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IP

[102] C. Ogino, N. Shibata, R. Sasai, K. Takaki, Y. Miyachi, S.I. Kuroda, et al., Construction of protein-modified TiO2 nanoparticles for use with ultrasound irradiation in a novel cell injuring method, Bioorganic Med. Chem. Lett. 20 (2010) 5320–5325. doi:10.1016/j.bmcl.2010.06.124.

NU

[103] K. Ninomiya, C. Ogino, S. Oshima, S. Sonoke, S.I. Kuroda, N. Shimizu, Targeted sonodynamic therapy using protein-modified TiO 2 nanoparticles, Ultrason. Sonochem. 19 (2012) 607–614. doi:10.1016/j.ultsonch.2011.09.009.

MA

[104] X. Zhang, Q. Zhang, Q. Peng, J. Zhou, L. Liao, X. Sun, et al., Hepatitis B virus preS1-derived lipopeptide functionalized liposomes for targeting of hepatic cells., Biomaterials. 35 (2014) 6130–41. doi:10.1016/j.biomaterials.2014.04.037.

D

[105] S. Hama, H. Akita, R. Ito, H. Mizuguchi, T. Hayakawa, H. Harashima,

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Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems, Mol. Ther. 13 (2006) 786–794. doi:10.1016/j.ymthe.2005.10.007.

AC

[106] M. Hyodo, Y. Sakurai, H. Akita, H. Harashima, ―Programmed packaging‖ for gene delivery., J. Control. Release. 193 (2014) 316–323. doi:10.1016/j.jconrel.2014.04.023. [107] M.N. Ara, T. Matsuda, M. Hyodo, Y. Sakurai, H. Hatakeyama, N. Ohga, et al., An aptamer ligand based liposomal nanocarrier system that targets tumor endothelial cells, Biomaterials. 35 (2014) 7110–7120. doi:10.1016/j.biomaterials.2014.04.087. [108] I.A. Khalil, K. Kogure, S. Futaki, S. Hama, H. Akita, M. Ueno, et al., Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery., Gene Ther. 14 (2007) 682–9. doi:10.1038/sj.gt.3302910. [109] K. Kusumoto, H. Akita, S. Santiwarangkool, H. Harashima, Advantages of ethanol dilution method for preparing GALA-modified liposomal siRNA carriers on the in vivo gene knockdown efficiency in pulmonary endothelium, Int. J. Pharm. 473 (2014) 144–147. doi:10.1016/j.ijpharm.2014.07.007. 35

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[110] E. Mastrobattista, M. a E.M. van der Aa, W.E. Hennink, D.J. a Crommelin, Artificial viruses: a nanotechnological approach to gene delivery., Nat. Rev. Drug Discov. 5 (2006) 115–121. doi:10.1038/nrd1960.

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[111] Y. Nishimura, H. Mieda, J. Ishii, C. Ogino, T. Fujiwara, A. Kondo, Targeting cancer cell-specific RNA interference by siRNA delivery using a complex carrier of affibody-displaying bio-nanocapsules and liposomes., J. Nanobiotechnology. 11 (2013) 19. doi:10.1186/1477-3155-11-19.

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Figure legends

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Fig. 1 Schematic diagram of HBV and a BNC. HBV virion (left) has three types of envelope protein (S, M, and L), lipid bilayer, HBcAg, polymerase, and genomic DNA.

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BNC (right) has L protein and lipid bilayer. The diameters of HBV and BNC are approx. 42 nm and approx. 100 nm, respectively.

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Fig. 2 Functional domains of L protein. Three types of functional domains are deployed outside of BNC. Human hepatocyte recognition domain (2-48 aa in L protein;

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within pre-S1 region); three fusogenic domains (9-24 aa (within pre-S1 region), 149-160 aa (within pre-S2 region), and 164-186 aa (within S region) in L protein), and polymerized-albumin receptor (PAR) domain (120-129 aa in L protein; within pre-S2

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region). Furthermore, at least three transmembrane domains reside in the S region that

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are necessary for the formation of BNCs. The numbers of the first amino acid residues of the pre-S1, pre-S2, and S regions, as well as the last residue of L protein, are

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indicated.

Fig. 3 Payload incorporation methods for BNCs.

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Fig. 4 BNC and its derivatives. Schematic representations and transmission electron micrographs are shown. (A) BNC, (B) liposomes, (C) BNC-liposome complex, and (D) virosome. Bars, 100 nm.

Fig. 5 Retargeting strategies for BNCs.

Fig. 6 Virus-inspired nanocarriers. (A) VLP, (B) virosome, and (C) virus-mimicking nanocarrier.

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Table 1. Pros and cons of the various payload incorporation methods

Electroporation

Nucleic acids, Drugsa

-

Genetic fusion

Proteins

+

Chemical conjugation

Proteins, Drugs

++

Liposome fusion

Proteins, Drugs, Nucleic acids, Beads

Virosome formation

Proteins, Drugs, Nucleic

Polymer fusion

Nucleic acids

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D TE CE P 38

++ -

++

++

++

++

++

+

Not limited to therapeutic compounds; includes general chemicals and fluorophores.

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a

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acids, Beads

Cytoplasmic delivery

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Mass scalability

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Methods

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Table 2. Drug and gene delivery by BNC technology Payloads

Size

In vitro

Human hepatic cells

Calcein/pDNA

210 nm

+

EGFRa

pDNAb

N.D.c

+

Human hepatic cells

pDNA

N.D.

Human liver tissue

Calcein

N.D.

Human hepatic cells

Calcein

N.D.

EGFR

b

[32]

+ (iv)

-

[33]

+ (iv)

-

[34]

Cys-substituted BNC e

e

[54]

N.D.

+

-

[57]

150 nm

+

Anti-EGFR Ab/ZZ

[73]

100-120 nm

+

-

[43]

+ (sc)d

ZZ

[58]

+ (ic)d

Anti-EGFR Ab/ZZ

[71]

Anti-EGFR

[82]

Fluorophore

conjugation

Not specified

Antigen

EGFR

Fluorophore

N.D.

+

EGFR

Fluorophore

90 nm

+

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Human hepatic cells

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EGF ligand/BNC

[72]

Chemical

50 nm

nanobody +

Octaarginine

[83]

Chlorotoxin

N.D

+

Chlorotoxin-Fc/ZZ

[80]

Fluorophore

70 nm

+

Anti-CD11c Ab/ZZ

[75]

β1-6 GlcNAc

Fluorophore

70 nm

+

+ (iv)

L4-PHA lectin

[81]

Human hepatic cells

pDNA/100-nm

200 nm

+

+ (iv)

-

[60]

+ (iv)

-

[61]

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Mouse DC

a

CE P

110 nm

MMP2a

fusion

[32]

Anti-EGFR Ab /ZZ

EGFP

Liposome

-

+ (iv)d

+

EGFR

Fluorophore

Ref.

N.D.

EGFP

Not specified

Notes

+

Human hepatic cells

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Genetic fusion

Taxol derivative

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Electroporation

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methods

In vivo

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Targets

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Incorporation

beads

Human hepatic cells

Doxorubicin

140 nm

+

Human hepatic cells

siRNA

160 nm

+

-

[62]

Human hepatic cells

miRNA

N.D.

+

-

[63]

Human hepatic cells

pDNA

130-240 nm

+

-

[65]

N.D.

+

-

[66]

Anti-CD11c Ab/ZZ

[75]

(PKA-driven) Human hepatic cells

pDNA (PKC-driven)

Mouse DC

Antigen

460 nm

+ (iv)

HER2

Calcein

100 nm

+

HER2-affibody

[78]

HER2

siRNA

80-120 nm

+

HER2-affibody

[111]

HER2

Calcein

100 nm

+

HER2-affibody

[85]

/GALA peptide β1-6 GlcNAc

pDNA

N.D. 39

+

L4-PHA lectin

[81]

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Virosome

Human hepatic cells

Doxorubicin

120 nm

+

Human hepatic cells

100-nm beads/

150-200 nm

+

200 nm

+

+ (iv)

With radiotherapy

[67]

formation

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[49]

a

Human hepatic cells

pDNA

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Polymer fusion

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gold nanoparticle

EGFR, epidermal growth factor receptor; MMP2, matrix metalloproteinase 2; DC, dendritic cells.

b

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pDNA, plasmid DNA; EGFP, enhanced green fluorescent protein.

c

N.D., not determined.

d

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IV, intravenously; SC, subcutaneously; IC, intracerebroventricularly.

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Ab, antibody; ZZ, ZZ-BNC.

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e

-

40

[70]

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Figure 1

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Figure 2

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Figure 3

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Figure 4

44

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D

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Figure 5

45

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Figure 6

CE P

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D

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