Progress of Endogenous and Exogenous Nanoparticles for Cancer Therapy and Diagnostics.

The focus of this brief review is to describe the application of nanoparticles, including endogenous nanoparticles (e.g., extracellular vesicles, EVs, and virus capsids) and exogenous nanoparticles (e.g., organic and inorganic materials) in cancer therapy and diagnostics. In this review, we mainly focused on EVs, where a recent study demonstrated that EVs secreted from cancer cells are associated with malignant alterations in cancer. EVs are expected to be used for cancer diagnostics by analyzing their informative cargo. Exogenous nanoparticles are also used in cancer diagnostics as imaging probes because they can be easily functionalized. Nanoparticles are promising targets for drug delivery system (DDS) development and have recently been actively studied. In this review, we introduce nanoparticles as a powerful tool in the field of cancer therapy and diagnostics and discuss issues and future prospects.

Reporter gene assay for membrane fusion of extracellular vesicles.

Extracellular vesicles (EVs) secreted by living cells are expected to deliver biological cargo molecules, including RNA and proteins, to the cytoplasm of recipient cells. There is an increasing need to understand the mechanism of intercellular cargo delivery by EVs. However, the lack of a feasible bioassay has hampered our understanding of the biological processes of EV uptake, membrane fusion, and cargo delivery to recipient cells. Here, we describe a reporter gene assay that can measure the membrane fusion efficiency of EVs during cargo delivery to recipient cells. When EVs containing tetracycline transactivator (tTA)-fused tetraspanins are internalized by recipient cells and fuse with cell membranes, the tTA domain is exposed to the cytoplasm and cleaved by tobacco etch virus protease to induce tetracycline responsive element (TRE)-mediated reporter gene expression in recipient cells. This assay (designated as EV-mediated tetraspanin-tTA delivery assay, ETTD assay), enabled us to assess the cytoplasmic cargo delivery efficiency of EVs in recipient cells. With the help of a vesicular stomatitis virus-derived membrane fusion protein, the ETTD assay could detect significant enhancement of cargo delivery efficiency of EVs. Furthermore, the ETTD assay could evaluate the effect of potential cargo delivery enhancers/inhibitors. Thus, the ETTD assay may contribute to a better understanding of the underlying mechanism of the cytoplasmic cargo delivery by EVs.

HBV Pre-S1-Derived Myristoylated Peptide (Myr47): Identification of the Inhibitory Activity on the Cellular Uptake of Lipid Nanoparticles.

The Myr47 lipopeptide, consisting of hepatitis B virus (HBV) pre-S1 domain (myristoylated 2-48 peptide), is an effective commercialized anti-HBV drug that prevents the interaction of HBV with sodium taurocholate cotransporting polypeptide (NTCP) on human hepatocytes, an activity which requires both N-myristoylation residue and specific amino acid sequences. We recently reported that Myr47 reduces the cellular uptake of HBV surface antigen (HBsAg, subviral particle of HBV) in the absence of NTCP expression. In this study, we analyzed how Myr47 reduces the cellular uptake of lipid nanoparticles (including liposomes (LPs) and HBsAg) without NTCP expression. By using Myr47 mutants lacking the HBV infection inhibitory activity, they could reduce the cellular uptake of LPs in an N-myristoylation-dependent manner and an amino acid sequence-independent manner, not only in human liver-derived cells but also in human non-liver-derived cells. Moreover, Myr47 and its mutants could reduce the interaction of LPs with apolipoprotein E3 (ApoE3) in an N-myristoylation-dependent manner regardless of their amino acid sequences. From these results, lipopeptides are generally anchored by inserting their myristoyl residue into the lipid bilayer and can inhibit the interaction of LPs/HBsAg with apolipoprotein, thereby reducing the cellular uptake of LPs/HBsAg. Similarly, Myr47 would interact with HBV, inhibiting the uptake of HBV into human hepatic cells, while the inhibitory effect of Myr47 may be secondary to its ability to protect against HBV infection.

Enhancing antibody-dependent cellular phagocytosis by Re-education of tumor-associated macrophages with resiquimod-encapsulated liposomes.

Tumor-associated macrophages (TAMs) exist in nearly all tumors, and form a major part of the tumor microenvironment. TAMs are divided into two groups: tumor-suppressing M1 type and tumor-promoting M2 type. Most TAMs are educated by the tumor cells to become M2 type, which support tumor growth and make immunotherapy ineffective. Antibody-dependent cellular phagocytosis (ADCP) is an important mechanism for antibody cancer therapy, and this mechanism is dependent on TAMs. In this study, we found that the M1 type macrophages elicit a more efficient ADCP response than the M2 type, which was confirmed by three tumor cell lines, Raji, A431, and SKBR3, along with their corresponding therapeutic antibody Rituximab, anti-EGFR mouse monoclonal antibody (clone 528), and Trastuzumab, respectively. Resiquimod (R848), an immune system activating agent, has been shown to stimulate the M1 type macrophages, and re-educate the TAMs from M2 type to M1 type. By treating TAMs with R848, the ADCP response increased significantly in vitro and in in vivo mouse xenograft models. R848 encapsulated liposomes (R848-LPs) not only accumulated efficiently in the tumor tissues, but also distributed in the TAMs. Synergizing the R848-LPs with the anti-EGFR mouse monoclonal antibody (clone 528) significantly inhibited WiDr-tumor growth in vivo. Our study also revealed that the TAM-targeted delivery of R848 is able to re-educate the TAMs to M1 type, enhance the ADCP effect of the antibodies, and hence, enhance the anti-tumor effect of the therapeutic 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.

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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: TAT-modified ZZ-BNC could be a useful GDS for uterine endometrial therapy via local uterine injection.

Induction of lipid droplets in non-macrophage cells as well as macrophages by liposomes and exosomes.

It has been reported that phospholipid nanoparticles (PNPs) including liposomes and exosomes could efficiently induce lipid droplets (LDs) in macrophages. However, in non-macrophage cells, the effects of PNPs on the induction of LDs have not been thoroughly investigated. In this report, we directly compared non-macrophage and macrophage cell lines in terms of LD induction by various formulation of liposomes containing phosphatidylserine and exosomes. All non-macrophage cell lines as well as macrophage cell lines tested in this study showed evident LD induction in response to these PNPs, though the efficacy of LD induction in non-macrophage cell lines varied considerably. Our results suggest that LD formation is a common and crucial response to PNPs in mammalian cells not only in macrophages but also in non-macrophage cells.

Oriented immobilization to nanoparticles enhanced the therapeutic efficacy of antibody drugs.

Antibody drugs have been important therapeutic agents for treating various diseases, such as cancer, rheumatism, and hypercholesterolemia, for the last three decades. Despite showing excellent therapeutic efficacy with good safety in vivo, they require high doses. We have developed a ∼30-nm bio-nanocapsule (ZZ-BNC) consisting of hepatitis B virus envelope L protein fused with the tandem form of protein A-derived IgG Fc-binding Z domain (ZZ-L protein), for tethering antibodies in an oriented immobilization manner. In this study, antibody drugs were spontaneously conjugated to ZZ-BNC, which displayed the IgG Fv regions outwardly. The anti-human epidermal growth factor receptor IgG conjugated to ZZ-BNC (α-hEGFR-ZZ-BNC) was endocytosed by the human epidermoid carcinoma A431 cells, with increases in cellular uptake by ∼1.5 fold, compared that of α-hEGFR IgG alone. The amount of α-hEGFR IgG in the late endosomes and lysosomes was increased from 4% to 33% by the conjugation to ZZ-BNC. The in vitro cytotoxicity of α-hEGFR-ZZ-BNC was higher by ∼10-fold than that of α-hEGFR IgG alone. Furthermore, in vivo tumor growth was significantly reduced by α-hEGFR-ZZ-BNC than by α-hEGFR IgG alone. Taken together, since endosomal EGFR, not cell surface EGFR, played a pivotal role in the EGFR-mediated signaling cascade, ZZ-BNC increased α-hEGFR IgG avidity by efficiently repressing the activation of hEGFR not only on the cell surface, but presumably also in the endosomes. These results strongly suggested that ZZ-BNC is a promising nano-scaffold for enhancing the therapeutic efficacy and reducing the dose of antibody drugs. STATEMENT OF SIGNIFICANCE: Antibody drugs are widely used for treating severe diseases, such as cancer, rheumatism, and hypercholesterolemia. These drugs are composed of naturally occurring biomaterials with low immunogenicity and toxicity, as well as long in vivo serum half-life. To achieve sufficient therapeutic efficacy, the dose of antibody drugs are unavoidably higher than those of conventional drugs. The present study shows an innovative way to reduce the dose of antibody drugs by using a nanocarrier-conjugated antibody. Oriented immobilization of the antibody enhanced its avidity, endocytosis efficiency, and therapeutic efficacy.

A hepatitis B virus-derived human hepatic cell-specific heparin-binding peptide: identification and application to a drug delivery system.

Viruses are naturally evolved nanocarriers that can evade host immune systems, attach specifically to the surfaces of target cells, enter the cells through endocytosis, escape from endosomes efficiently, and then transfer their genomes to host cells. Hepatitis B virus (HBV) is a ∼42 nm enveloped DNA virus that can specifically infect human hepatic cells. To utilize the HBV-derived early infection machinery in synthetic nanocarriers, the human hepatic cell-binding site (i.e., the sodium taurocholate co-transporting polypeptide (NTCP)-binding site, with myristoylated pre-S1(2-47)) and the low pH-dependent fusogenic domain (pre-S1(9-24)) are indispensable for targeting and endosomal escape, respectively. However, cell-surface NTCP has recently been shown not to be involved in the initial attachment of HBV. In this study, we identified a novel heparin-binding site (pre-S1(30-42)) in the N-terminal half of the pre-S1 region, which presumably interacts with cell-surface heparan sulfate proteoglycan (HSPG) and plays a pivotal role in the initial attachment of HBV to human hepatic cells. The evolutionarily conserved amino acid residues Asp-31, Trp-32, and Asp-33 are indispensable for the heparin-binding activity. Liposomes (LPs) displaying the peptide were endocytosed by human hepatic cells in a cell-surface heparin-dependent manner and delivered doxorubicin to human hepatic cells more efficiently than myristoylated pre-S1(2-47)-displaying LPs. These results demonstrated that the pre-S1(30-42) peptide is the most promising HBV-derived targeting peptide for synthetic nanocarriers, and that this peptide exhibits high specificity for human hepatic cells and efficiently induces endocytosis.

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

BACKGROUND: Various 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). RESULTS: After 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. CONCLUSION: The 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 SIGNIFICANCE: We 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.

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.

Cellular uptake of hepatitis B virus envelope L particles is independent of sodium taurocholate cotransporting polypeptide, but dependent on heparan sulfate proteoglycan.

Sodium taurocholate cotransporting polypeptide (NTCP) was recently discovered as a hepatitis B virus (HBV) receptor, however, the detailed mechanism of HBV entry is not yet fully understood. We investigated the cellular entry pathway of HBV using recombinant HBV surface antigen L protein particles (bio-nanocapsules, BNCs). After the modification of L protein in BNCs with myristoyl group, myristoylated BNCs (Myr-BNCs) were found to bind to NTCP in vitro, and inhibit in vitro HBV infection competitively, suggesting that Myr-BNCs share NTCP-dependent infection machinery with HBV. Nevertheless, the cellular entry rates of Myr-BNCs and plasma-derived HBV surface antigen (HBsAg) particles were the same as those of BNCs in NTCP-overexpressing HepG2 cells. Moreover, the cellular entry of these particles was mainly driven by heparan sulfate proteoglycan-mediated endocytosis regardless of NTCP expression. Taken together, cell-surface NTCP may not be involved in the cellular uptake of HBV, while presumably intracellular NTCP plays a critical role.

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

As drug delivery systems, nanocarriers should be capable of executing the following functions: evasion of the host immune system, targeting to the diseased site, 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 hepatocyte-targeting domain, fusogenic domain, and polymerized-albumin receptor domain of HBV envelope L protein on its surface-by overexpressing the L protein in 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 with liposomes, or chemical modification. In this review, we describe BNC-related technology, discuss retargeting strategies for BNCs, and outline other virus-inspired nanocarriers.

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.

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.

Targeting of polyplex to human hepatic cells by bio-nanocapsules, hepatitis B virus surface antigen L protein particles.

We have previously demonstrated that lipoplex, a complex of cationic liposomes and DNA, could be targeted to human hepatic cells in vitro and in vivo by conjugation with bio-nanocapsules (BNCs) comprising hepatitis B virus (HBV) surface antigen L protein particles. Because the BNC-lipoplex complexes were endowed with the human hepatic cell-specific infection machinery from HBV, the complexes showed excellent specific transfection efficiency in human hepatic cells. In this study, we have found that polyplex (a complex of polyethyleneimine (PEI) and DNA) could form stable complexes with BNCs spontaneously. The diameter and ζ-potential of BNC-polyplex complexes are about 240 nm and +3.54 mV, respectively, which make them more suitable for in vivo use than polyplex alone. BNC-polyplex complexes with an N/P ratio (the molar ratio of the amine group of PEI to the phosphate group of DNA) of 40 showed excellent transfection efficiency in human hepatic cells. When acidification of endosomes was inhibited by bafilomycin A1, the complexes showed higher transfection efficiency than polyplex itself, strongly suggesting that the complexes escaped from endosomes by both fusogenic activity of BNCs and proton sponge activity of polyplex. Furthermore, the cytotoxicity is comparable to that of polyplex of the same N/P value. Thus, BNC-polyplex complexes would be a promising gene delivery carrier for human liver-specific gene therapy.