Bio-nanocapsule Research Articles

Binding of Nanoparticles Harboring Recombinant Large Surface Protein of Hepatitis B Virus to Scavenger Receptor Class B Type 1.

(1) Background: As nanoparticles containing the hepatitis B virus (HBV) large (L) surface protein produced in yeast are expected to be useful as a carrier for targeting hepatocytes, they are also referred to as bio-nanocapsules (BNCs). However, a definitive cell membrane receptor for BNC binding has not yet been identified. (2) Methods: By utilizing fluorescence-labeled BNCs, we examined BNC binding to the scavenger receptor class B type 1 (SR-B1) expressed in HEK293T cells. (3) Results: Analyses employing SR-B1 siRNA and expression of SR-B1 fused with a green fluorescent protein (SR-B1-GFP) indicated that BNCs bind to SR-B1. As mutagenesis induced in the SR-B1 extracellular domain abrogates or attenuates BNC binding and endocytosis via SR-B1 in HEK293T cells, it was suggested that the ligand-binding site of SR-B1 is similar or close among high-density lipoprotein (HDL), silica, liposomes, and BNCs. On the other hand, L protein was suggested to attenuate an interaction between phospholipids and SR-B1. (4) Conclusions: SR-B1 can function as a receptor for binding and endocytosis of BNCs in HEK293T cells. Being expressed various types of cells, it is suggested that functions as a receptor for BNCs not only in HEK293T cells but also in other types of cells.

Polymerized Albumin Receptor of Hepatitis B Virus for Evading the Reticuloendothelial System.

Various strategies, such as optimization of surface chemistry, size, shape, and charge, have been undertaken to develop nanoparticles (NPs) as DDS (drug delivery system) nanocarriers for evading the reticuloendothelial system (RES) in vivo. We previously developed a hollow NP composed of hepatitis B virus (HBV) surface antigen L proteins and lipid bilayers, hereinafter referred to as bio-nanocapsule (BNC), as a nonviral DDS nanocarrier. Such a BNC harbors the HBV-derived human hepatic cell-specific infection mechanism, and intravenously injected BNCs by themselves were shown to avoid clearance by RES-rich organs and accumulate in target tissues. In this study, since the surface modification with albumins is known to prolong the circulation time of nanomedicines, we examined whether the polymerized albumin receptor (PAR) of BNCs contributes to RES evasion in mouse liver. Our results show that NPs conjugated with peptides possessing sufficient PAR activity were captured by Kupffer cells less efficiently in vitro and were able to circulate for a longer period of time in vivo. Comparing with polyethylene glycol, PAR peptides were shown to reduce the recognition by RES to equal content. Taken together, our results strongly suggest that the PAR domain of BNCs, as well as HBV, harbors an innate RES evasion mechanism. Therefore, the surface modification with PAR peptides could be an alternative strategy for improving the pharmacodynamics and pharmacokinetics of forthcoming nanomedicines.

Enhanced sugar chain detection by oriented immobilization of Fc-fused lectins.

We report a novel scaffold for clustering and oriented immobilization of human IgG1 Fc-fused lectins on biosensors without chemical modifications. This approach uses a bio-nanocapsule (BNC) displaying a tandem form of IgG Fc-binding Z domains derived from Staphylococcus aureus protein A (ZZ-BNC). Incorporating ZZ-BNC effectively increased both the sensitivity and sugar chain-binding capacity compared with the condition without ZZ-BNC.

Mechanism of affinity-enhanced protein adsorption on bio-nanocapsules studied by viscoelasticity measurement with wireless QCM biosensor

Recent advances in functionalized bio-nanocapsules (BNCs) have allowed a significant sensitivity enhancement in label-free biosensors. It is suggested that sensitivity amplification is caused by the higher binding affinity of BNCs to target antibodies. We here study the high-affinity interaction mechanism between BNCs and antibodies with wireless high-frequency QCM biosensors. We first confirmed the higher affinity between BNCs and human immunoglobulin G. We then found that the number of binding sites for the target antibody significantly increases by immobilizing BNC molecules on the sensor surface. We finally studied changes in viscoelasticity near the sensor surface using a MEMS wireless QCM biosensor using up to the ninth overtone (522 MHz). The inversely determined effective shear modulus on BNCs was found to be significantly lower than that on a standard surface on which the receptor molecules were immobilized. We have thus clarified that this surface flexibility achieves high affinity with target antibodies.

DDS Nanocarriers Mimicking Early Infection Machinery of Viruses

Viruses are natural nanocarriers that deliver various biological cargos, such as DNA, RNA, and proteins. We are developing a new nanocarrier by mimicking the early mechanism of infection by hepatitis B virus (HBV). When the HBV envelope L protein is overexpressed in yeast cells, hollow nanoparticles displaying L proteins are synthesized. This nanoparticle, namely a bio-nanocapsule (BNC), can specifically attach to, and then internalize into, human hepatic cells by implementing the early mechanism of infection by HBV. In this review, we outlined the cellular uptake mechanism of HBV/BNC linking to L protein function. The L protein contains several functional domains in the pre-S1 region, including the fusogenic domain and the heparin-binding domain. The fusogenic domain corresponding to the pre-S1(9-24) region is responsible for the low pH-dependent membrane fusion of BNC. The heparin-binding domain corresponding to the pre-S1(30-42) region has a strong affinity to heparin as compared to that of known heparin-binding peptides, such as vitronectin and gp120 in human immunodeficiency virus-1. This heparin-binding domain binds to heparan sulfate proteoglycan (HSPG) at the cell surface of human hepatic cells. These functional domains are present in any virus, thus, these viral envelope proteins are very useful in designing novel DDS nanocarriers.

In vivo uterine local gene delivery system using TAT-displaying bionanocapsules.

The uterus is an organ that is directly accessible via the transvaginal route, whereas the drug delivery system and the gene delivery system (GDS) for the uterus are very limited, even in animal models. In the present study, we optimized a bionanocapsule (BNC) comprising a hepatitis B virus envelope L-protein particle, for which a structurally similar particle has been used as an immunogen of a conventional HB vaccine worldwide for more than 30 years, as a local uterine GDS using a mouse model. To display various antibodies for re-targeting to different cells other than hepatic cells, the pre-S1 region of BNC was replaced with a tandem form of the protein A-derived immunoglobulin G Fc-interacting region (Z domain, ZZ-BNC). To induce strong cell adhesion after local administration into the uterine cavity, ZZ-BNC was modified with a transactivator of transcription (TAT) peptide. Gene transfer using TAT-modified ZZ-BNC is approximately 5000- or 18-fold more efficient than the introduction of the same dose of naked DNAs or the use of the cationic liposomes, respectively. TAT-modified ZZ-BNC was rapidly eliminated from the uterus and had no effect on the pregnancy rate, litter size or fetal growth. TAT-modified ZZ-BNC could be a useful GDS for uterine endometrial therapy via local uterine injection.

Construction of a Macrophage-Targeting Bio-nanocapsule-Based Nanocarrier.

The construction protocol of bio-nanocapsule (BNC)-based nanocarriers, named GL-BNC and GL-virosome, for targeted drug delivery to macrophages is described here. First, genes encoding the Streptococcus sp. protein G-derived C2 domain (binds to IgG Fc) and Finegoldia magna protein L-derived B1 domain (binds to Igκ light chain) are prepared by PCR amplification. Subsequently, the genes encoding hepatic cell-specific binding domain of hepatitis B virus envelope L protein are replaced by these PCR products. The expression plasmid for this fused gene (encoding GL-fused L protein) can be used to transform Saccharomyces cerevisiae AH22R- cells. To obtain GL-BNC, the transformed yeast cells are disrupted with glass beads, treated with heat, and then subjected to IgG affinity column chromatography followed by size exclusion column chromatography. In addition, GL-BNCs can be fused with liposomes to form GL-virosome. The targeted delivery of GL-BNC and GL-virosome to macrophages can be confirmed by in vitro phagocytosis assays using the murine macrophage cell line RAW264.7.

Ultrahigh-sensitive wireless QCM with bio-nanocapsules

Quartz-crystal-microbalance (QCM) biosensor is a mass-sensitive biosensor, and its sensitivity in detecting target proteins can be improved with weighted detection antibodies. In this communication, we propose a mass-amplified detection of biomarkers by bio-nanocapsules (BNCs) in wireless high-frequency multichannel QCM biosensor. BNC exhibits molecular mass of 6.57 MDa and possesses many immunoglobulin-G (IgG) Fc-binding Z domains on its surface, making it possible to display ∼60 antibodies in an oriented immobilization manner. We find that by reinforcing the bond strength between the Z domain and the Fc part of antibody with the cross-linker, a high sensitive detection of biomarkers with QCM is realized. We used multichanel wireless QCM system with fundamental resonance frequencies above 54 MHz and performed detection of C-reactive protein. The detection limit is lower than 10 pg/mL, confirming that combination of BNC and wireless high-frequency QCM allows high sensitivity detection of biomarkers.

CD11c-specific bio-nanocapsule enhances vaccine immunogenicity by targeting immune cells

BackgroundVarious nanocarriers have been used to deliver subunit vaccines specifically to dendritic cells (DCs) for the improvement of immunogenicity. However, due to their insufficient DC priming ability, these vaccines could not elicit effective innate immunity. We have recently developed a DC-targeting bio-nanocapsule (BNC) by displaying anti-CD11c IgGs via protein A-derived IgG Fc-binding Z domain on the hepatitis B virus envelope L protein particles (α-DC-ZZ-BNC).ResultsAfter the chemical modification with antigens (Ags), the α-DC-ZZ-BNC-Ag complex could deliver Ags to DCs efficiently, leading to effective DC maturation and efficient endosomal escape of Ags, followed by Ag-specific T cell responses and IgG productions. Moreover, the α-DC-ZZ-BNC modified with Japanese encephalitis virus (JEV) envelope-derived D3 Ags could confer protection against 50-fold lethal dose of JEV injection on mice.ConclusionThe α-DC-ZZ-BNC-Ag platform was shown to induce humoral and cellular immunities effectively without any adjuvant.

Development of a macrophage-targeting and phagocytosis-inducing bio-nanocapsule-based nanocarrier for drug delivery

Macrophage hyperfunction or dysfunction is tightly associated with various diseases, such as osteoporosis, inflammatory disorder, and cancers. However, nearly all conventional drug delivery system (DDS) nanocarriers utilize endocytosis for entering target cells; thus, the development of macrophage-targeting and phagocytosis-inducing DDS nanocarriers for treating these diseases is required. In this study, we developed a hepatitis B virus (HBV) envelope L particle (i.e., bio-nanocapsule (BNC)) outwardly displaying a tandem form of protein G-derived IgG Fc-binding domain and protein L-derived IgG Fab-binding domain (GL-BNC). When conjugated with the macrophage-targeting ligand, mouse IgG2a (mIgG2a), the GL-BNC itself, and the liposome-fused GL-BNC (i.e., GL-virosome) spontaneously initiated aggregation by bridging between the Fc-binding domain and Fab-binding domain with mIgG2a. The aggregates were efficiently taken up by macrophages, whereas this was inhibited by latrunculin B, a phagocytosis-specific inhibitor. The mIgG2a-GL-virosome containing doxorubicin exhibited higher cytotoxicity toward macrophages than conventional liposomes and other BNC-based virosomes. Thus, GL-BNCs and GL-virosomes may constitute promising macrophage-targeting and phagocytosis-inducing DDS nanocarriers. Statement of SignificanceWe have developed a novel macrophage-targeting and phagocytosis-inducing bio-nanocapsule (BNC)-based nanocarrier named GL-BNC, which comprises a hepatitis B virus envelope L particle outwardly displaying protein G-derived IgG Fc- and protein L-derived IgG Fab-binding domains in tandem. The GL-BNC alone or liposome-fused form (GL-virosomes) could spontaneously aggregate when conjugated with macrophage-targeting IgGs, inducing phagocytosis by the interaction between IgG Fc of aggregates and FcγR on phagocytes. Thereby these aggregates were efficiently taken up by macrophages. GL-virosomes containing doxorubicin exhibited higher cytotoxicity towards macrophages than ZZ-virosomes and liposomes. Our results suggested that GL-BNCs and GL-virosomes would serve as promising drug delivery system nanocarriers for targeting delivery to macrophages.

Low immunogenic bio-nanocapsule based on hepatitis B virus escape mutants

Bio-nanocapsules (BNCs) consisting of hepatitis B virus surface antigen (HBsAg) L proteins and phospholipids are used as efficient non-viral carriers for liver-specific delivery of genes and drugs. Considering the administration to HB vaccinees and HB patients, endogenous anti-HBsAg immunoglobulins (HBIGs) may reduce the delivery efficacy and prevent repetitive administration. Therefore, low immunogenic BNCs were generated by inserting two point mutations in the HBsAg L protein, which were found in HBV escape mutants. Escape mutant-type BNC (emBNC) showed 50% lower HBIG binding capacity than that of parental BNC (wtBNC). It induced HBIG production to a lesser extent than that associated with wtBNC in BALB/c mice. The emBNC could accumulate into human hepatocyte-derived tumor in mice pre-treated with HBIGs. The complex of emBNC and cationic liposomes could deliver plasmid DNA to HepG2 cells efficiently in the presence of HBIGs. Thus, emBNC could evade HBIG-neutralizing antibodies, expanding the clinical utility of BNC-based nanomedicine.

Preclinical evaluation of cisplatin-incorporated bio-nanocapsules as chemo-radiotherapy for human hepatocellular carcinoma.

The incidence of hepatocellular carcinoma (HCC) has continued to increase worldwide, and advanced HCC is difficult to treat using the currently available therapeutics. Chemoradiotherapy with cisplatin (cis-diamminedichloroplatinum, CDDP) is expected to confer a curative benefit on HCC patients; however, its application is limited due to side-effects such as acute nephrotoxicity as well as the conventionally limited application of chemoradiotherapy for HCC. For the practical application of this drug in the clinical setting, we formulated a novel drug carrier-comprising bio-nanocapsule (BNC) and liposomal CDDP (BNC-LP-CDDP) that recognizes the human liver and releases CDDP. BNC-LP-CDDP showed selectively high cytotoxicity for HCC cells, and markedly reduced the survival fractions of HCC when combined with ionizing radiation (IR) treatment in invitro assays. In particular, the treatment of mice bearing human HCC with BNC-LP-CDDP and 3Gy IR showed 95.68% growth inhibition, whereas IR treatment alone showed 65.6% growth inhibition. Moreover, BNC-LP-CDDP led to the withdrawal of CDDP-induced nephrotoxicity. These results indicate that BNC-LP-CDDP in combination with IR markedly enhanced the chemo-radiotherapeutic efficacy and eliminated CDDP induced nephrotoxicity, thus, suggesting the potential for its clinical application as human HCC therapy.

Affibody-displaying bio-nanocapsules effective in EGFR, typical biomarker, expressed in various cancer cells

The expression of epidermal growth factor receptor (EGFR) across a wide range of tumor cells has attracted attention for use as a tumor marker in drug delivery systems. Therefore, binding molecules with the ability to target EGFR have been developed. Among them, we focused on affibodies that are binding proteins derived from staphylococcal protein A. By displaying affibody (ZEGFR) binding to EGFR on the surface of a bio-nanocapsule (BNC) derived from a hepatitis B virus (HBV), we developed an altered BNC (ZEGFR-BNC) with a high specificity to EGFR-expressing cells. We considered two different types of ZEGFR (Z955 and Z1907), and found that the Z1907 dimer-displaying BNC ([Z1907]2-BNC) could effectively bind to EGFR-expressing cells and deliver drugs to the cytosol. Since this study showed that [Z1907]2-BNC could target EGFR-expressing cells, we would use this particle as a drug delivery carrier for various cancer cells expressing EGFR.

Core-fucosylation plays a pivotal role in hepatitis B pseudo virus infection: a possible implication for HBV glycotherapy.

The functions of cell surface proteins, such as growth factor receptors and virus/bacteria-entry receptors, can be dynamically regulated by oligosaccharide modifications. In the present study, we investigated the involvement of glycosylation in hepatitis B virus (HBV) entry into hepatoma cells. Infection of oligosaccharide-remodeling hepatoma cells with a pseudo virus of HBV, bio-nanocapsule (BNC), was evaluated by flow cytometry and confocal microscopy. Among various experiments using several hepatoma cells, marked difference was observed between Huh6 cells and HB611 cells, which were established by HBV gene transfection into hepatoma cells. Comprehensive oligosaccharide analysis showed dramatic increases of core fucosylation in HB611 cells, compared with Huh6 cells. Knock down of fucosyltransferase 8 (FUT8) reduced BNC entry into HB611 cells. In contrast, overexpression of FUT8 in Huh6 cells increased BNC entry. Although expression of sodium taurocholate cotransporting polypeptide (NTCP), which is one of HBV receptors was very similar between Huh6 and HB611 cells, proteins coprecipitated with NTCP were dependent on levels of core-fucosylation, suggesting that core-fucosylation regulates BNC entry into hepatoma cells. Our findings demonstrate that core-fucosylation is an important glycosylation for HBV infection of hepatoma cells through HBV-receptor-mediated endocytosis. Down-regulation of core-fucosylation may be a novel target for HBV therapy.

Mutational analysis of hepatitis B virus pre-S1 (9–24) fusogenic peptide

A hollow nanoparticle known as a bio-nanocapsule (BNC) consisting of hepatitis B virus (HBV) envelope L protein and liposome (LP) can encapsulate drugs and genes and thereby deliver them in vitro and in vivo to human hepatic tissues, specifically by utilizing the HBV-derived infection machinery. Recently, we identified a low pH-dependent fusogenic domain at the N-terminal part of the pre-S1 region of the HBV L protein (amino acid residues 9 to 24; NPLGFFPDHQLDPAFG), which shows membrane destabilizing activity (i.e., membrane fusion, membrane disruption, and payload release) upon interaction with target LPs. In this study, instead of BNC and HBV, we generated LPs displaying a mutated form of the pre-S1 (9–24) peptide, and performed a membrane disruption assay using target LPs containing pyranine (fluorophore) and p-xylene-bis (N-pyridinium bromide) (DPX) as a quencher. The membrane disruption activity was found to correlate with the hydrophobicity of the whole structure, while the peptide retained a random-coil structure even under low pH condition. One large hydrophobic cluster (I) and one small hydrophobic cluster (II) residing in the peptide would be connected by the protonation of residues D16 and D20, and thereby exhibit strong membrane disruption activity in a low pH-dependent manner. Furthermore, the introduction of a positively charged residue enhanced the activity significantly, suggesting that a sole positively charged residue (H17) may be important for the interaction with target LPs by electrostatic interaction. Collectively, these results suggest that the pre-S1 (9–24) peptide may be involved in the endosomal escape of the BNC's payloads, as well as in the HBV uncoating process.

Bio-nanocapsule-based scaffold improves the sensitivity and ligand-binding capacity of mammalian receptors on the sensor chip.

Mammalian receptors are recognized as target molecules for drug discovery, and chemical libraries have been screened for both potential antagonists and agonists mainly by ligand-binding assays using immobilized receptors. A bio-nanocapsule (BNC) of approximately 30 nm that displays a tandem form of the protein A-derived immunoglobulin G (IgG) Fc-binding Z domains (denoted as ZZ-BNC) has been developed for both clustering and oriented immobilization of IgGs on the solid phase of immunosensors. In this study, human IgG1 Fc-fused vascular endothelial growth factor (VEGF) receptor was immobilized through ZZ-BNC on the sensor chip of quartz crystal microbalance (ZZ-BNC-coating). When compared with direct adsorption and protein A-coating, the sensor chip showed higher sensitivity (∽46- and ∽165-fold, respectively) and larger ligand-binding capacity (∽4- and ∽18-fold, respectively). Furthermore, the number of VEGF molecules bound to its receptor increased from 0.20 (direct adsorption) to 2.06 by ZZ-BNC-coating, strongly suggesting that ZZ-BNC reduced the steric hindrance near ligand recognition sites through oriented immobilization. Similarly, the sensitivity and ligand-binding capacity of leptin and prolactin receptors were both enhanced at a level comparable to that observed for the VEGF receptor. Thus, the combination of ZZ-BNC and Fc-fused receptors could significantly improve the function of ligand-binding assays.

Bio-nanocapsules displaying various immunoglobulins as an active targeting-based drug delivery system

The bio-nanocapsule (BNC) is an approximately 30-nm particle comprising the hepatitis B virus (HBV) envelope L protein and a lipid bilayer. The L protein harbors the HBV-derived infection machinery; therefore, BNC can encapsulate payloads such as drugs, nucleic acids, and proteins and deliver them into human hepatocytes specifically in vitro and in vivo. To diversify the possible functions of BNC, we generated ZZ-BNC by replacing the domain indispensable for the human hepatotrophic property of BNC (N-terminal region of L protein) with the tandem form of the IgG Fc-binding Z domain of Staphylococcus aureus protein A. Thus, the ZZ-BNC is an active targeting-based drug delivery system (DDS) nanocarrier that depends on the specificity of the IgGs displayed. However, the Z domain limits the animal species and subtypes of IgGs that can be displayed on ZZ-BNC. In this study, we introduced into BNC an Ig κ light chain-binding B1 domain of Finegoldia magna protein L (protein-L B1 domain) and an Ig Fc-binding C2 domain of Streptococcus species protein G (protein-G C2 domain) to produce LG-BNC. The LL-BNC was constructed in a similar way using a tandem form of the protein-L B1 domain. Both LG-BNC and LL-BNC could display rat IgGs, mouse IgG1, human IgG3, and human IgM, all of which not binding to ZZ-BNC, and accumulate in target cells in an antibody specificity-dependent manner. Thus, these BNCs could display a broad spectrum of Igs, significantly improving the prospects for BNCs as active targeting-based DDS nanocarriers. Statement of SignificanceWe previously reported that ZZ-BNC, bio-nanocapsule deploying the IgG-binding Z domain of protein A, could display cell-specific antibody in an oriented immobilization manner, and act as an active targeting-based DDS nanocarrier. Since the Z domain can only bind to limited types of Igs, we generated BNCs deploying other Ig-binding domains: LL-BNC harboring the tandem form of Ig-binding domain of protein L, and LG-BNC harboring the Ig binding domains of protein L and protein G sequentially. Both BNCs could display a broader spectrum of Igs than does the ZZ-BNC. When these BNCs displayed anti-CD11c IgG or anti-EGFR IgG, both of which cannot bind to Z domain, they could bind to and then enter their respective target cells.

Elucidation of the early infection machinery of hepatitis B virus by using bio-nanocapsule.

Currently, hepatitis B virus (HBV), upon attaching to human hepatocytes, is considered to interact first with heparan sulfate proteoglycan (HSPG) via an antigenic loop of HBV envelope S protein. Then, it is promptly transferred to the sodium taurocholate cotransporting polypeptide (NTCP) via the myristoylated N-terminal sequence of pre-S1 region (from Gly-2 to Gly-48, HBV genotype D), and it finally enters the cell by endocytosis. However, it is not clear how HSPG passes HBV to NTCP and how NTCP contributes to the cellular entry of HBV. Owing to the poor availability and the difficulty of manipulations, including fluorophore encapsulation, it has been nearly impossible to perform biochemical and cytochemical analyses using a substantial amount of HBV. A bio-nanocapsule (BNC), which is a hollow nanoparticle consisting of HBV envelope L protein, was efficiently synthesized in Saccharomyces cerevisiae. Since BNC could encapsulate payloads (drugs, genes, proteins) and specifically enter human hepatic cells utilizing HBV-derived infection machinery, it could be used as a model of HBV infection to elucidate the early infection machinery. Recently, it was demonstrated that the N-terminal sequence of pre-S1 region (from Asn-9 to Gly-24) possesses low pH-dependent fusogenic activity, which might play a crucial role in the endosomal escape of BNC payloads and in the uncoating process of HBV. In this minireview, we describe a model in which each domain of the HBV L protein contributes to attachment onto human hepatic cells through HSPG, initiation of endocytosis, interaction with NTCP in endosomes, and consequent provocation of membrane fusion followed by endosomal escape.

Intracellular trafficking of bio-nanocapsule–liposome complex: Identification of fusogenic activity in the pre-S1 region of hepatitis B virus surface antigen L protein

Bio-nanocapsules (BNCs) are a hollow nanoparticle consisting of about 100-nm liposome (LP) embedding about 110 molecules of hepatitis B virus (HBV) surface antigen (HBsAg) L protein as a transmembrane protein. Owing to the human hepatocyte-recognizing domains on the N-terminal region (pre-S1 region), BNCs have recently been shown to attach and enter into human hepatic cells using the early infection mechanism of HBV. Since BNCs could form a complex with an LP containing various drugs and genes, BNC–LP complexes have been used as a human hepatic cell-specific drug and gene-delivery system in vitro and in vivo. However, the role of BNCs in cell entry and intracellular trafficking of payloads in BNC–LP complexes has not been fully elucidated. In this study, we demonstrate that low pH-dependent fusogenic activity resides in the N-terminal part of pre-S1 region (NPLGFFPDHQLDPAFG), of which the first FF residues are essential for the activity, and which facilitates membrane fusion between LPs in vitro. Moreover, BNC–LP complexes can bind human hepatic cells specifically, enter into the cells via clathrin-mediated endocytosis, and release their payloads mostly into the cytoplasm. Taken together, the BNC portion of BNC–LP complexes can induce membrane fusion between LPs and endosomal membranes under low pH conditions, and thereby facilitate the endosomal escape of payloads. Furthermore, the fusogenic domain of the pre-S1 region of HBsAg L protein may play a pivotal role in the intracellular trafficking of not only BNC–LP complexes but also of HBV.

Virosomes of hepatitis B virus envelope L proteins containing doxorubicin: synergistic enhancement of human liver-specific antitumor growth activity by radiotherapy.

Bionanocapsules (BNCs) are hollow nanoparticles consisting of hepatitis B virus (HBV) envelope L proteins and have been shown to deliver drugs and genes specifically to human hepatic tissues by utilizing HBV-derived infection machinery. The complex of BNCs with liposomes (LPs), the BNC–LP complexes (a LP surrounded by BNCs in a rugged spherical form), could also become active targeting nanocarriers by the BNC function. In this study, under acidic conditions and high temperature, BNCs were found to fully fuse with LPs (smooth-surfaced spherical form), deploying L proteins with a membrane topology similar to that of BNCs (ie, virosomes displaying L proteins). Doxorubicin (DOX) was efficiently encapsulated via the remote loading method at 14.2%±1.0% of total lipid weight (mean ± SD, n=3), with a capsule size of 118.2±4.7 nm and a ζ-potential of −51.1±1.0 mV (mean ± SD, n=5). When mammalian cells were exposed to the virosomes, the virosomes showed strong cytotoxicity in human hepatic cells (target cells of BNCs), but not in human colon cancer cells (nontarget cells of BNCs), whereas LPs containing DOX and DOXOVES (structurally stabilized PEGylated LPs containing DOX) did not show strong cytotoxicity in either cell type. Furthermore, the virosomes preferentially delivered DOX to the nuclei of human hepatic cells. Xenograft mice harboring either target or nontarget cell-derived tumors were injected twice intravenously with the virosomes containing DOX at a low dose (2.3 mg/kg as DOX, 5 days interval). The growth of target cell-derived tumors was retarded effectively and specifically. Next, the combination of high dose (10.0 mg/kg as DOX, once) with tumor-specific radiotherapy (3 Gy, once after 2 hours) exhibited the most effective antitumor growth activity in mice harboring target cell-derived tumors. These results demonstrated that the HBV-based virosomes containing DOX could be an effective antitumor nanomedicine specific to human hepatic tissues, especially in combination with radiotherapy.