Computational design of non-porous pH-responsive antibody nanoparticles.

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and is important for targeted delivery of biologics. Here we describe the design of octahedral non-porous nanoparticles with a targeting antibody on the two-fold symmetry axis, a designed trimer programmed to disassemble below a tunable pH transition point on the three-fold axis, and a designed tetramer on the four-fold symmetry axis. Designed non-covalent interfaces guide cooperative nanoparticle assembly from independently purified components, and a cryo-EM density map closely matches the computational design model. The designed nanoparticles can package protein and nucleic acid payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between 5.9 and 6.7. The ability to incorporate almost any antibody into a non-porous pH-dependent nanoparticle opens up new routes to antibody-directed targeted delivery.

Computational design of non-porous, pH-responsive antibody nanoparticles.

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and important for targeted delivery of biologics. We describe the design of octahedral non-porous nanoparticles with the three symmetry axes (four-fold, three-fold, and two-fold) occupied by three distinct protein homooligomers: a de novo designed tetramer, an antibody of interest, and a designed trimer programmed to disassemble below a tunable pH transition point. The nanoparticles assemble cooperatively from independently purified components, and a cryo-EM density map reveals that the structure is very close to the computational design model. The designed nanoparticles can package a variety of molecular payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between to 5.9-6.7. To our knowledge, these are the first designed nanoparticles with more than two structural components and with finely tunable environmental sensitivity, and they provide new routes to antibody-directed targeted delivery.

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.

Engineering of Extracellular Vesicles for Small Molecule-Regulated Cargo Loading and Cytoplasmic Delivery of Bioactive Proteins.

Cytoplasmic delivery of functional proteins into target cells remains challenging for many biological agents to exert their therapeutic effects. Extracellular vesicles (EVs) are expected to be a promising platform for protein delivery; however, efficient loading of proteins of interest (POIs) into EVs remains elusive. In this study, we utilized small compound-induced heterodimerization between FK506 binding protein (FKBP) and FKBP12-rapamycin-binding (FRB) domain to sort bioactive proteins into EVs using the FRB-FKBP system. When CD81, a typical EV marker protein, and POI were fused with FKBP and FRB, respectively, rapamycin induced the binding of these proteins through the FKBP-FRB interaction and recruited the POIs into EVs. The released EVs, displaying the virus-derived membrane fusion protein, delivered the POI cargo into recipient cells and their functionality in the recipient cells was confirmed. Furthermore, we demonstrated that CD81 could be replaced with other EV-enriched proteins, such as CD63 or HIV Gag. Thus, the FRB-FKBP system enables the delivery of functional proteins and paves the way for EV-based protein delivery platforms.

Recent advances in animal cell technologies for industrial and medical applications.

The industrial use of living organisms for bioproduction of valued substances has been accomplished mostly using microorganisms. To produce high-value bioproducts such as antibodies that require glycosylation modification for better performance, animal cells have been recently gaining attention in bioengineering because microorganisms are unsuitable for producing such substances. Furthermore, animal cells are now classified as products because a large number of cells are required for use in regenerative medicine. In this article, we review animal cell technologies and the use of animal cells, focusing on useable cell generation and large-scale production of animal cells. We review recent advance in mammalian cell line development because this is the first step in the production of recombinant proteins, and it largely affects the efficacy of the production. We next review genetic engineering technology focusing on CRISPR-Cas system as well as surrounding technologies as these methods have been gaining increasing attention in areas that use animal cells. We further review technologies relating to bioreactors used in the context of animal cells because they are essential for the mass production of target products. We also review tissue engineering technology because tissue engineering is one of the main exits for mass-produced cells; in combination with genetic engineering technology, it can prove to be a promising treatment for patients with genetic diseases after the establishment of induced pluripotent stem cell technology. The technologies highlighted in this review cover brief outline of the recent animal cell technologies related to industrial and medical applications.

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.

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.

Real-Time Luminescence Assay for Cytoplasmic Cargo Delivery of Extracellular Vesicles.

Extracellular vesicles (EVs) have been considered to deliver biological cargos between cells and mediate intercellular communication and potential drug delivery carriers. However, the mechanisms that underlie the biological process of EV uptake and cytoplasmic cargo release in recipient cells are largely unknown. Quantitative and real-time assays for the assessment of cargo delivery efficiency inside recipient cells have not been feasible. In this study, we developed an EV cargo delivery (EVCD) assay using a split luciferase called a NanoBiT system. Recipient cells expressing LgBiT, a large subunit of luciferase, emit luminescence when EV cargo proteins fused with a small luminescence tag (HiBiT tag) that can complement LgBiT are delivered to the cytoplasm of recipient cells. Using the EVCD assay, the cargo delivery efficiency of EVs could be quantitatively measured in real time. This assay was highly sensitive in detecting a single event of cargo delivery per cell. We found that modification of EVs with a virus-derived fusogenic protein significantly enhanced the cytoplasmic cargo delivery; however, in the absence of a fusogenic protein, the cargo delivery efficiency of EVs was below the threshold of the assay. The EVCD assay could assess the effect of entry inhibitors on EV cargo delivery. Furthermore, using a luminescence microscope, the cytoplasmic cargo delivery of EVs was directly visualized in living cells. This assay could reveal the biological mechanism of the cargo delivery processes of EVs.

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.

A regulatory role of scavenger receptor class B type 1 in endocytosis and lipid droplet formation induced by liposomes containing phosphatidylethanolamine in HEK293T cells.

We have recently reported that phosphatidylethanolamine (PE)-containing liposomes are endocytosed and then induce lipid droplets (LDs) in HEK293T cells. In this study, we elucidated a mechanism responsible for endocytosis of PE-containing liposomes and induction of LDs. By using fluorescence-labeled liposomes and flow cytometry, we found that PE-containing liposomes were very efficiently internalized in HEK293T cells. However, Block lipid transporter-1 (BLT-1) only marginally suppressed the uptake of these liposomes, indicating that entire liposomes were mostly taken up in these cells. They were therefore inferred to express abundant PE receptors responsible for endocytosis of PE-containing liposomes. We examined the expression of 52 candidate genes through transcriptomic analyses and eventually narrowed it down to four candidate genes, which were abundantly expressed in HEK293T cells. Among siRNAs targeting these candidates, scavenger receptor class B type 1 (SR-B1) siRNA showed the most profound reduction in PE liposomal uptake. Conversely, the expression of SR-B1 by transfection of an expression plasmid enhanced the uptake of PE-containing liposomes. After the internalization of PE-containing liposomes, they were colocalized with endosomes/lysosomes and SR-B1, which indicates that these liposomes are taken up in HEK293T cells at least partially through the endosomal/lysosomal pathway. A specific anti-SR-B1-antibody blocked the uptake of PE-containing liposomes in HEK293T cells while LD formation in these cells induced by PE-containing liposomes was suppressed by treatment with SR-B1 siRNA. These results demonstrate that SR-B1 functions as a receptor for the endocytosis of PE-containing liposomes and regulates the formation of LDs induced by PE-containing liposomes in HEK293T cells.

Where does the cargo go?: Solutions to provide experimental support for the "extracellular vesicle cargo transfer hypothesis".

It is widely believed that extracellular vesicles (EVs) mediate intercellular communications by functioning as messengers. EVs contain various biomolecules, including nucleic acids and proteins, as cargo in the internal space. Thus, it has been postulated that this cargo can be transferred from donor cells to recipient cells, leading to phenotypic changes in the recipient cells. However, there is a lack of experimental evidence for the aforementioned hypothesis, that EVs function as messengers. This is presumably because of a lack of rigorous methodologies for EV research. Although cells usually incorporate nanoparticles (NPs) from the extracellular space via endocytosis, these NPs are processed through the endo/lysosomal system and do not escape to the cytoplasm unless they disrupt or fuse with the endo/lysosomal membrane. Whether EVs actually are capable of escaping endo/lysosomes is still debatable. In contrast, viruses have evolved to efficiently deliver their cargo (viral proteins and genetic material) into the cytoplasm of host (recipient) cells by circumventing endo/lysosomal degradation. Thus, it may be helpful to compare EVs to viruses in terms of cargo delivery. The present technological issues that hinder obtaining support for the "EV cargo transfer hypothesis" are summarized and potential solutions for EV research are proposed.

[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.