Electrical stimulation facilitates NADPH production in pentose phosphate pathway and exerts an anti-inflammatory effect in macrophages.

Macrophages play an important role as effector cells in innate immune system. Meanwhile, macrophages activated in a pro-inflammatory direction alter intracellular metabolism and damage intact tissues by increasing reactive oxygen species (ROS). Electrical stimulation (ES), a predominant physical agent to control metabolism in cells and tissues, has been reported to exert anti-inflammatory effect on immune cells. However, the mechanism underlying the anti-inflammatory effects by ES is unknown. This study aimed to investigate the effect of ES on metabolism in glycolytic-tricarboxylic acid cycle (TCA) cycle and inflammatory responses in macrophages. ES was performed on bone marrow-derived macrophages and followed by a stimulation with LPS. The inflammatory cytokine expression levels were analyzed by real-time polymerase chain reaction and ELISA. ROS production was analyzed by CellRox Green Reagent and metabolites by capillary electrophoresis-mass spectrometry. As a result, ES significantly reduced proinflammatory cytokine expression levels and ROS generation compared to the LPS group and increased glucose-1-phosphate, a metabolite of glycogen. ES also increased intermediate metabolites of the pentose phosphate pathway (PPP); ribulose-5-phosphate, rebose-5 phosphate, and nicotinamide adenine dinucleotide phosphate, a key factor of cellular antioxidation systems, as well as α-Ketoglutarate, an anti-oxidative metabolite in the TCA cycle. Our findings imply that ES enhanced NADPH production with enhancement of PPP, and also decreased oxidative stress and inflammatory responses in macrophages.

Skeletal myotube-derived extracellular vesicles enhance itaconate production and attenuate inflammatory responses of macrophages.

Introduction: Macrophages play an important role in the innate immunity. While macrophage inflammation is necessary for biological defense, it must be appropriately controlled. Extracellular vesicles (EVs) are small vesicles released from all types of cells and play a central role in intercellular communication. Skeletal muscle has been suggested to release anti-inflammatory factors, but the effect of myotube-derived EVs on macrophages is unknown. As an anti-inflammatory mechanism of macrophages, the immune responsive gene 1 (IRG1)-itaconate pathway is essential. In this study, we show that skeletal muscle-derived EVs suppress macrophage inflammatory responses, upregulating the IRG1-itaconate pathway. Methods: C2C12 myoblasts were differentiated into myotubes and EVs were extracted by ultracentrifugation. Skeletal myotube-derived EVs were administered to mouse bone marrow-derived macrophages, then lipopolysaccharide (LPS) stimulation was performed and inflammatory cytokine expression was measured by RT-qPCR. Metabolite abundance in macrophages after addition of EVs was measured by CE/MS, and IRG1 expression was measured by RT-PCR. Furthermore, RNA-seq analysis was performed on macrophages after EV treatment. Results: EVs attenuated the expression of LPS-induced pro-inflammatory factors in macrophages. Itaconate abundance and IRG1 expression were significantly increased in the EV-treated group. RNA-seq analysis revealed activation of the PI3K-Akt and JAK-STAT pathways in macrophages after EV treatment. The most abundant miRNA in myotube EVs was miR-206-3p, followed by miR-378a-3p, miR-30d-5p, and miR-21a-5p. Discussion: Skeletal myotube EVs are supposed to increase the production of itaconate via upregulation of IRG1 expression and exhibited an anti-inflammatory effect in macrophages. This anti-inflammatory effect was suggested to involve the PI3K-Akt and JAK-STAT pathways. The miRNA profiles within EVs implied that miR-206-3p, miR-378a-3p, miR-30d-5p, and miR-21a-5p may be responsible for the anti-inflammatory effects of the EVs. In summary, in this study we showed that myotube-derived EVs prevent macrophage inflammatory responses by activating the IRG1-itaconate pathway.

Fluorophore-Decorated Mie Resonant Silicon Nanosphere for Scattering/Fluorescence Dual-Mode Imaging.

Inorganic nanoparticles with multiple functions have been attracting attention as multimodal nanoprobes in bioimaging, biomolecule detection, and medical diagnosis and treatment. A drawback of conventional metallic nanoparticle-based nanoprobes is the Ohmic losses that lead to fluorescence quenching of attached molecules and local heating under light irradiation. Here, metal-free nanoprobes capable of scattering/fluorescence dual-mode imaging are developed. The nanoprobes are composed of a silicon nanosphere core having efficient Mie scattering in the visible to near infrared range and a fluorophore doped silica shell. The dark-field scattering and photoluminescence images/spectra for nanoprobes made from different size silicon nanospheres and different kinds of fluorophores are studied by single particle spectroscopy. The fluorescence spectra are strongly modified by the Mie modes of a silicon nanosphere core. By comparing scattering and fluorescence spectra and calculated Purcell factors, the fluorescence enhancement factor is quantitatively discussed. In vitro scattering/fluorescence imaging studies on human cancer cells demonstrate that the developed nanoparticles work as scattering/fluorescence dual-mode imaging nanoprobes.

In vivo stealthified molecularly imprinted polymer nanogels incorporated with gold nanoparticles for radiation therapy.

Radiation therapy is a representative therapeutic approach for cancer treatment, wherein the development of efficient radiation sensitizers with low side effects is critical. In this study, a novel stealth radiation sensitizer based on Au-embedded molecularly imprinted polymer nanogels (Au MIP-NGs) was developed for low-dose X-ray radiation therapy. Surface plasmon resonance measurements reveal the good affinity and selectivity of the obtained Au MIP-NGs toward the target dysopsonic protein, human serum albumin. The protein recognition capability of the nanogels led to the formation of the albumin-rich protein corona in the plasma. The Au MIP-NGs acquire stealth capability in vivo through protein corona regulation using the intrinsic dysopsonic proteins. The injection of Au MIP-NGs improved the efficiency of the radiation therapy in mouse models of pancreatic cancer. The growth of the pancreatic tumor was inhibited even at low X-ray doses (2 Gy). The novel strategy reported in this study for the synthesis of stealth nanomaterials based on nanomaterial-protein interaction control shows significant potential for application even in other approaches for cancer treatment, diagnostics, and theranostics. This strategy paves a way for the development of a wide range of effective nanomedicines for cancer therapy.

Reactive oxygen species-inducing titanium peroxide nanoparticles as promising radiosensitizers for eliminating pancreatic cancer stem cells.

BACKGROUND: Despite recent advances in radiotherapy, radioresistance in patients with pancreatic cancer remains a crucial dilemma for clinical treatment. Cancer stem cells (CSCs) represent a major factor in radioresistance. Developing a potent radiosensitizer may be a novel candidate for the eradication of pancreatic CSCs. METHODS: CSCs were isolated from MIA PaCa-2 and PANC1 human pancreatic cancer cell lines. Titanium peroxide nanoparticles (TiOxNPs) were synthesized from titanium dioxide nanoparticles (TiO2NPs) and utilized as radiosensitizers when added one hour prior to radiation exposure. The antitumor activity of this novel therapeutic strategy was evaluated against well-established pancreatic CSCs model both in vitro and in vivo. RESULTS: It is shown that TiOxNPs combined with ionizing radiation exhibit anti-cancer effects on radioresistant CSCs both in vitro and in vivo. TiOxNPs exhibited a synergistic effect with radiation on pancreatic CSC-enriched spheres by downregulating self-renewal regulatory factors and CSC surface markers. Moreover, combined treatment suppressed epithelial-mesenchymal transition, migration, and invasion properties in primary and aggressive pancreatic cancer cells by reducing the expression of proteins relevant to these processes. Notably, radiosensitizing TiOxNPs suppressed the growth of pancreatic xenografts following primary or dissociating sphere MIA PaCa-2 cell implantation. It is inferred that synergy is formed by generating intolerable levels of reactive oxygen species (ROS) and inactivating the AKT signaling pathway. CONCLUSIONS: Our data suggested the use of TiOxNPs in combination with radiation may be considered an attractive therapeutic strategy to eliminate pancreatic CSCs.

Biocompatible polymer-modified gold nanocomposites of different shapes as radiation sensitizers.

Radiation therapy is a powerful approach for cancer treatment due to its low invasiveness. The development of radiation sensitizers is of great importance as they assist in providing radiation therapy at a low dose. In this study, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-modified gold nanocomposites of different shapes were created using the grafting-to approach to serve as a novel radiation sensitizer with high cellular uptake. The effect of the shape of the nanocomposite on cellular uptake by the breast cancer cell line MCF-7 was also investigated. The PMPC-modified gold nanostars showed the highest cellular uptake compared to the other gold nanocomposites (spheres and rods), whereas cell cytotoxicity was negligible among all candidates. Furthermore, the therapeutic effect of radiation of PMPC-modified nanostars was the highest among all the gold nanocomposites. These results clearly indicate that the shape of the gold nanocomposite is an important parameter for cellular uptake and radiation sensitizing effects in breast cancer cells.

Titanium oxide nano-radiosensitizers for hydrogen peroxide delivery into cancer cells.

Polyacrylic acid-modified titanium peroxide nanoparticles (PAA-TiOx NPs) are promising radiosensitizers that enhance the therapeutic effect of X-ray irradiation after local injection into tumors. However, the mechanism for this reaction has remained unclear with the exception of the involvement of hydrogen peroxide (H2O2), which is released by PAA-TiOx NPs to a liquid phase during dispersion. In the present study, a clonogenic assay was used to compare PAA-TiOx NPs with free H2O2 molecules to investigate the effect exerted on the radiosensitivity of cancer cells in vitro. A cell-free dialysis method revealed that a portion of the H2O2 adsorbed onto the PAA-TiOx NPs during synthesis could be released during a treatment regimen. The H2O2 release lasted for 7 h, which was sufficient for one radiation treatment procedure. For in vitro experiments, cultured human pancreatic cancer cells took up PAA-TiOx NPs in 10 min after administration. Interestingly, when the cells were washed with a buffer after treatment with either a PAA-TiOx NP or H2O2 solution, the intracellular H2O2 levels remained higher with PAA-TiOx NP treatment compared with the H2O2 solution treatment. Furthermore, the effects of subsequent X-ray irradiation corresponded to the intracellular H2O2 levels. These results indicate that PAA-TiOx NPs are efficient carriers of H2O2 into cancer cells and thus enhance the radiosensitivity.

A Comparative Assessment of Mechanisms and Effectiveness of Radiosensitization by Titanium Peroxide and Gold Nanoparticles.

The development of potentially safe radiosensitizing agents is essential to enhance the treatment outcomes of radioresistant cancers. The titanium peroxide nanoparticle (TiOxNP) was originally produced using the titanium dioxide nanoparticle, and it showed excellent reactive oxygen species (ROS) generation in response to ionizing radiation. Surface coating the TiOxNPs with polyacrylic acid (PAA) showed low toxicity to the living body and excellent radiosensitizing effect on cancer cells. Herein, we evaluated the mechanism of radiosensitization by PAA-TiOxNPs in comparison with gold nanoparticles (AuNPs) which represent high-atomic-number nanoparticles that show a radiosensitizing effect through the emission of secondary electrons. The anticancer effects of both nanoparticles were compared by induction of apoptosis, colony-forming assay, and the inhibition of tumor growth. PAA-TiOxNPs showed a significantly more radiosensitizing effect than that of AuNPs. A comparison of the types and amounts of ROS generated showed that hydrogen peroxide generation by PAA-TiOxNPs was the major factor that contributed to the nanoparticle radiosensitization. Importantly, PAA-TiOxNPs were generally nontoxic to healthy mice and caused no histological abnormalities in the liver, kidney, lung, and heart tissues.

In Vivo Evaluation of the ZHER2-BNC/LP Carrier Encapsulating an Anticancer Drug and a Radiosensitizer.

Radiosensitizing therapy for cancer treatment that enhances the effect of existing radiation therapy and enables noninvasive therapy has attracted attention. In this study, to achieve target cell-specific noninvasive cancer treatment using a ZHER2-bionanocapsule/liposome (BNC/LP), a carrier that binds to human epidermal growth factor receptor 2 (HER2), we evaluated the delivery of anticancer drugs and radiosensitizers and treatment effects in vitro and in vivo in mice. Target cell-specific cytotoxic activity and antitumor effects were confirmed following delivery of doxorubicin-encapsulated particles. In addition, cell damage due to radiosensitizing effects was confirmed in combination with X-ray irradiation following delivery of particles containing polyacrylic acid-modified titanium peroxide nanoparticles as a radiosensitizer. Furthermore, even when the particles were injected via the tail vein of mice, they accumulated in the tumor and exhibited an antitumor effect because of radiosensitization. Therefore, ZHER2-BNC/LP is expected to be a carrier that releases small-molecule drugs into the target cell cytoplasm and delivers a radiosensitizer such as inorganic nanoparticles, enabling combination therapy with X-rays to the target tumor.

Metabolic engineering of the 2-ketobutyrate biosynthetic pathway for 1-propanol production in Saccharomyces cerevisiae.

BACKGROUND: To produce 1-propanol as a potential biofuel, metabolic engineering of microorganisms, such as E. coli, has been studied. However, 1-propanol production using metabolically engineered Saccharomyces cerevisiae, which has an amazing ability to produce ethanol and is thus alcohol-tolerant, has infrequently been reported. Therefore, in this study, we aimed to engineer S. cerevisiae strains capable of producing 1-propanol at high levels. RESULTS: We found that the activity of endogenous 2-keto acid decarboxylase and alcohol/aldehyde dehydrogenase is sufficient to convert 2-ketobutyrate (2 KB) to 500 mg/L 1-propanol in yeast. Production of 1-propanol could be increased by: (i) the construction of an artificial 2 KB biosynthetic pathway from pyruvate via citramalate (cimA); (ii) overexpression of threonine dehydratase (tdcB); (iii) enhancement of threonine biosynthesis from aspartate (thrA, thrB and thrC); and (iv) deletion of the GLY1 gene that regulates a competing pathway converting threonine to glycine. With high-density anaerobic fermentation of the engineered S. cerevisiae strain YG5C4231, we succeeded in producing 180 mg/L 1-propanol from glucose. CONCLUSION: These results indicate that the engineering of a citramalate-mediated pathway as a production method for 1-propanol in S. cerevisiae is effective. Although optimization of the carbon flux in S. cerevisiae is necessary to harness this pathway, it is a promising candidate for the large-scale production of 1-propanol.

In vivo tissue distribution and safety of polyacrylic acid-modified titanium peroxide nanoparticles as novel radiosensitizers.

Polyacrylic acid (PAA)-modified titanium peroxide nanoparticles (PAA-TiOx NPs) are promising radiosensitizers. PAA-TiOx NPs were synthesized from commercial TiO2 nanoparticles that were modified with PAA and functionalized by H2O2 treatment. To realize practical clinical uses for PAA-TiOx NPs, their tissue distribution and acute toxicity were evaluated using healthy mice and mice bearing tumors derived from xenografted MIAPaCa-2 human pancreatic cancer cells. Healthy mice were injected with PAA-TiOx NPs at 25 mg/kg body weight via the tail vein, and tumor-bearing mice were injected either into the tumor locally or via the tail vein. The concentration of PAA-TiOx NPs in major organs was determined over time using inductively coupled-plasma atomic emission spectrometry. After 1 h, 12% of the PAA-TiOx NP dose had accumulated in the tumor, and 2.8% of the dose remained after 1 week. Such high accumulation could be associated with enhanced permeability and retention effects of the tumor, as PAA-TiOx NPs are composed of inorganic particles and polymers, without tumor-targeting molecules. The liver accumulated the largest proportion of the injected nanoparticles, up to 42% in tumor-bearing mice. Blood biochemical parameters were also investigated after intravenous injection of PAA-TiOx NPs in healthy mice. PAA-TiOx NPs invoked a slight change in various liver-related biochemical parameters, but no liver injury was observed over the practical dose range. In the future, PAA-TiOx NPs should be modified to prevent accumulation in the liver and minimize risk to patients.

Engineering Human Epidermal Growth Receptor 2-Targeting Hepatitis B Virus Core Nanoparticles for siRNA Delivery in Vitro and in Vivo.

Hepatitis B virus core (HBc) particles acquire the capacity to disassemble and reassemble in a controlled manner, allowing entrapment and delivery of drugs and macromolecules to cells. HBc particles are made of 180-240 copies of 21 kDa protein monomers, assembled into 30-34 nm diameter icosahedral particles. In this study, we aimed at formulating HBc particles for the delivery of siRNA for gene silencing in vitro and in vivo. We have previously reported recombinant HBc particles expressing ZHER2 affibodies, specifically targeting human epidermal growth receptor 2 (HER2)-expressing cancer cells (ZHER2-ΔHBc). siRNA was encapsulated within the ZHER2-ΔHBc particles following disassembly and reassembly. The ZHER2-ΔHBc-siRNA hybrids were able to secure the encapsulated siRNA from serum and nucleases in vitro. Enhanced siRNA uptake in HER2-expressing cancer cells treated with ZHER2-ΔHBc-siRNA hybrids was observed compared to the nontargeted HBc-siRNA hybrids in a time- and dose-dependent manner. A successful in vitro polo-like kinase 1 (PLK1) gene knockdown was demonstrated in cancer cells treated with ZHER2-ΔHBc-siPLK1 hybrids, to levels comparable to commercial transfecting reagents. Interestingly, ZHER2-ΔHBc particles exhibit intrinsic capability of reducing the solid tumor mass, independent of siPLK1 therapy, in an intraperitoneal tumor model following intraperitoneal injection.

Engineering hepatitis B virus core particles for targeting HER2 receptors in vitro and in vivo.

Hepatitis B Virus core (HBc) particles have been studied for their potential as drug delivery vehicles for cancer therapy. HBc particles are hollow nano-particles of 30-34 nm diameter and 7 nm thick envelopes, consisting of 180-240 units of 21 kDa core monomers. They have the capacity to assemble/dis-assemble in a controlled manner allowing encapsulation of various drugs and other biomolecules. Moreover, other functional motifs, i.e. receptors, receptor binding sequences, peptides and proteins can be expressed. This study focuses on the development of genetically modified HBc particles to specifically recognise and target human epidermal growth factor receptor-2 (HER2)-expressing cancer cells, in vitro and in vivo, for future cancer therapy. The non-specific binding capacity of wild type HBc particles was reduced by genetic deletion of the sequence encoding arginine-rich domains. A specific HER2-targeting was achieved by expressing the ZHER2 affibodies on the HBc particles surface. In vitro studies showed specific uptake of ZHER2-ΔHBc particles in HER2 expressing cancer cells. In vivo studies confirmed positive uptake of ZHER2-ΔHBc particles in HER2-expressing tumours, compared to non-targeted ΔHBc particles in intraperitoneal tumour-bearing mice models. The present results highlight the potential of these nanocarriers in targeting HER2-positive metastatic abdominal cancer following intra-peritoneal administration.

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.

Characterization of titanium dioxide nanoparticles modified with polyacrylic acid and H2O2 for use as a novel radiosensitizer.

An induction of polyacrylic acid-modified titanium dioxide with hydrogen peroxide nanoparticles (PAA-TiO2/H2O2 NPs) to a tumor exerted a therapeutic enhancement of X-ray irradiation in our previous study. To understand the mechanism of the radiosensitizing effect of PAA-TiO2/H2O2 NPs, analytical observations that included DLS, FE-SEM, FT-IR, XAFS, and Raman spectrometry were performed. In addition, highly reactive oxygen species (hROS) which PAA-TiO2/H2O2 NPs produced with X-ray irradiation were quantified by using a chemiluminescence method and a EPR spin-trapping method. We found that PAA-TiO2/H2O2 NPs have almost the same characteristics as PAA-TiO2. Surprisingly, there were no significant differences in hROS generation. However, the existence of H2O2 was confirmed in PAA-TiO2/H2O2 NPs, because spontaneous hROS production was observed w/o X-ray irradiation. In addition, PAA-TiO2/H2O2 NPs had a curious characteristic whereby they absorbed H2O2 molecules and released them gradually into a liquid phase. Based on these results, the H2O2 was continuously released from PAA-TiO2/H2O2 NPs, and then released H2O2 assumed to be functioned indirectly as a radiosensitizing factor.

Titanium peroxide nanoparticles enhanced cytotoxic effects of X-ray irradiation against pancreatic cancer model through reactive oxygen species generation in vitro and in vivo.

BACKGROUND: Biological applications of nanoparticles are rapidly increasing, which introduces new possibilities to improve the efficacy of radiotherapy. Here, we synthesized titanium peroxide nanoparticles (TiOxNPs) and investigated their efficacy as novel agents that can potently enhance the effects of radiation in the treatment of pancreatic cancer. METHODS: TiOxNPs and polyacrylic acid-modified TiOxNPs (PAA-TiOxNPs) were synthesized from anatase-type titanium dioxide nanoparticles (TiO2NPs). The size and morphology of the PAA-TiOxNPs was evaluated using transmission electron microscopy and dynamic light scattering. The crystalline structures of the TiO2NPs and PAA-TiOxNPs with and without X-ray irradiation were analyzed using X-ray absorption. The ability of TiOxNPs and PAA-TiOxNPs to produce reactive oxygen species in response to X-ray irradiation was evaluated in a cell-free system and confirmed by flow cytometric analysis in vitro. DNA damage after X-ray exposure with or without PAA-TiOxNPs was assessed by immunohistochemical analysis of γ-H2AX foci formation in vitro and in vivo. Cytotoxicity was evaluated by a colony forming assay in vitro. Xenografts were prepared using human pancreatic cancer MIAPaCa-2 cells and used to evaluate the inhibition of tumor growth caused by X-ray exposure, PAA-TiOxNPs, and the combination of the two. RESULTS: The core structures of the PAA-TiOxNPs were found to be of the anatase type. The TiOxNPs and PAA-TiOxNPs showed a distinct ability to produce hydroxyl radicals in response to X-ray irradiation in a dose- and concentration-dependent manner, whereas the TiO2NPs did not. At the highest concentration of TiOxNPs, the amount of hydroxyl radicals increased by >8.5-fold following treatment with 30 Gy of radiation. The absorption of PAA-TiOxNPs enhanced DNA damage and resulted in higher cytotoxicity in response to X-ray irradiation in vitro. The combination of the PAA-TiOxNPs and X-ray irradiation induced significantly stronger tumor growth inhibition compared to treatment with either PAA-TiOxNPs or X-ray alone (p < 0.05). No apparent toxicity or weight loss was observed for 43 days after irradiation. CONCLUSIONS: TiOxNPs are potential agents for enhancing the effects of radiation on pancreatic cancer and act via hydroxyl radical production; owing to this ability, they can be used for pancreatic cancer therapy in the future.

Mutation of arginine residues to avoid non-specific cellular uptakes for hepatitis B virus core particles.

BACKGROUND: The hepatitis B virus core (HBc) particle is known as a promising new carrier for the delivery of drugs and nucleic acids. However, since the arginine-rich domain that is located in the C-terminal region of the HBc monomer binds to the heparan sulphate proteoglycan on the cell surface due to its positive charge, HBc particles are introduced non-specifically into a wide range of cells. To avoid non-specific cellular uptake with the intent to control the ability of cell targeting, we individually replaced the respective arginine (R) residues of the arginine-rich domain located in amino acid positions 150-159 in glycine (G) residues. RESULTS: The mutated HBc particles in which R154 was replaced with glycine (G) residue (R154G) showed a drastic decrease in the ability to bind to the heparan sulphate proteoglycan and to avoid non-specific cellular uptake by several types of cancer cells. CONCLUSIONS: Because this mutant particle retains most of its C-terminal arginine-rich residues, it would be useful in the targeting of specificity-altered HBc particles in the delivery of nucleic acids.

A display of pH-sensitive fusogenic GALA peptide facilitates endosomal escape from a Bio-nanocapsule via an endocytic uptake pathway.

BACKGROUND: An affibody-displaying bio-nanocapsule (ZHER2-BNC) with a hepatocyte specificity derived from hepatitis B virus (HBV) was converted into an affibody, ZHER2, that recognizes HER2 receptors. This affibody was previously reported to be the result of the endocytosis-dependent specific uptake of proteins and siRNA into target cancer cells. To assist the endosomal escape of inclusions, a helper lipid with pH-sensitive fusogenic ability (1,2-dioleoyl-sn-glycero-3-phos phoethanolamine; DOPE) was conjugated with a ZHER2-BNC. FINDINGS: In this study, we displayed a pH-sensitive fusogenic GALA peptide on the surface of a particle in order to confer the ability of endosomal escape to a ZHER2-BNC. A GALA-displaying ZHER2-BNC purified from yeast uneventfully formed a particle structure. Furthermore, endosomal escape of the particle was facilitated after endocytic uptake and release of the inclusions to the cytoplasm without the cell toxicity. CONCLUSION: The genetic fusion of a GALA peptide to the virus-like particle confers the ability of endosomal escape.

Genetic engineering of bio-nanoparticles for drug delivery: a review.

Techniques using nanotechnology in the detection and treatment of cancers have made great progress in multidisciplinary fields. The advances in drug delivery systems (DDSs) have been supported mainly by the development of varied nanoparticles (NPs). Although the NPs based on organic and inorganic materials are integral parts in DDSs, bio-nanoparticles containing biopolymer and virus-like particles (VLPs) are attractive biomaterials for DDSs because of their unique features originating in bio-based materials, such as biocompatibility, biodegradability and low immunogenicity. It is notable that these NPs additionally have a great advantage to enable the easy and flexible alteration of their features by genetic engineering approaches. Controlling the sequence and oligomeric process of polypeptide genes permits a variety of choices in type or size of biopolymeric NPs (e.g., elastin-like polypeptide NPs). In contrast, the functional genes are often inserted into the coding sequences for self-assembled proteins to give the VLPs (e.g., hemagglutinating virus of Japan, adeno-associated virus, human immunodeficiency virus-1, simian virus 40 and hepatitis B virus) additional functions. Thus, genetic engineering readily allow alterations of the properties of NPs (e.g., particle shape, size and stability) and grant of new abilities (e.g., cell-specificity and drug loading and release). In this review, we introduce recent advances in bio-nanoparticles from the standpoint of engineering.

Targeting cancer cell-specific RNA interference by siRNA delivery using a complex carrier of affibody-displaying bio-nanocapsules and liposomes.

BACKGROUND: Small interfering RNA (siRNA) has attracted attention in the field of nucleic acid medicine as a RNA interference (RNAi) application that leads to gene silencing due to specific messenger RNA (mRNA) destruction. However, since siRNA is unstable in blood and unable to cross the cell membrane, encapsulation of siRNA into a carrier is required. RESULTS: In this study, we used a carrier that combined ZHER2-displaying bio-nanocapsule (derived from hepatitis B virus surface antigen) and liposomes in a complex in order to investigate the feasibility of effective and target-cell-specific RNAi applications. As a result, by observing RNAi only in HER2-expressing breast cancer cells, using our proposed methodology, we successfully demonstrated target-cell-specific delivery and effective function expression of siRNA. CONCLUSIONS: These findings show that, in the field of nucleic acid medicine, ZHER2-BNC/LP can be a useful carrier for siRNA delivery, and could also become a useful tool for gene silencing and to accomplish protein knock-down.