APCChE 2019

Hall L, am, Day 2, Tuesday Abstracts

Session 7. Biochemical engineering

[SC]PL201 Fabrication of a breathing bronchial-like platform for pulmonary drug application
Ying-ru CHEN, Jen-huang HUANG
National Tsing Hua University, Hsinchu, Taiwan

In recent years, researchers have shown an increase interest in lung disease owning to worse air pollution globally and high pulmonary attention. Several studies have examined issues related to the development of pulmonary administration of drug delivery. Yet, they still face several problems with the effectiveness of preclinical predictions on human drug responses and the shortage of high fidelity in organ-level cell testing without breathing simulation in vitro. The aim of this study was to fabricate a live and simple bronchial-like monitor with breathing motion mechanism for pulmonary drug testing. This bronchial-like monitor is regulated by deformation of the elastic membrane, leading to spontaneously air flow passing in and out. The deformation of membrane is dominated by different diameters of microchannel embedded in the bronchial-like monitor. The restriction of microchannel is contributed from the liquid flow controlled by a peristaltic pump. Therefore, the breathing mechanism can be manipulated by liquid flow rate and channel size. When liquid flow rate sets to be 0.84 ml/min (shear stress is 0.15 dyn/cm2), 328 ホシl/min of air flow rate and 0.49 dyn/cm2 of shear stress can be generated in the airway channel of the monitor. Additionally, the pattern of pressure drop in the airway channel also shows periodic tendency as successfully mimicking breathing motion of human. The Reynold number notes to be 0.30 that fully supports the laminar air flow in the bronchial-like airway. Furthermore, we cultured lung carcinoma A549 cells in the monitor with cyclic air flow to recapitulate the physiological relevant bronchial-like breathing model. This model, in the future, can be utilized to reconstruct the human healthy bronchiolar epithelium and to observe the toxicity and efficacy of pulmonary drug with breathing motion mechanism.

[SC]PL202 Mucoadhesive Solid-in-Oil nanodispersions for intranasal vaccination
1 Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Fukuoka City, Fukuoka, Japan
2 Advanced Transdermal Drug Delivery System Center, Kyushu University, Fukuoka City, Fukuoka, Japan
3 Center for Future Chemistry, Kyushu University, Fukuoka City, Fukuoka, Japan

The intranasal delivery of vaccines has recently emerged as an attractive alternative to injection. The main mechanism of the intranasal vaccination is that the antigens in the vaccine transport via M cells in the nasal epithelium, then resident dendritic cells capture antigens and process them to induce the immune responses. However, it shows several disadvantages: short residence time of the antigens in nasal cavity due to the rapid nasal mucociliary clearance and weak immune responses due to the inefficient antigens transportation to the immune system.
To overcome these issues, a Solid-in-Oil (S/O) nanodispersion technique was developed for intranasal vaccine delivery. It is based on nanodispersions of hydrophilic antigens coated with hydrophobic surfactant molecules in an oil vehicle. Surfactant molecules and the oil vehicle could increase the nasal residence time of the antigens due to its mucoadhesive effect. Moreover, nanosized particles is expected to improve the delivery efficiency of the antigens to the immune system and induce strong immune responses (Fig.1).
We investigated the potential of intranasal vaccination by using S/O nanodispersions loaded with a model antigen ovalbumin (OVA). S/O nanodispersions containing OVA in different oil vehicles were prepared and the formulation using squalene oil was chosen due to the small particle size. Moreover, nasal absorption and nasal residence time of OVA were assessed to be increased by S/O nanodispersions. Finally, the OVA-specific antibody levels from serum and nasal mucosal washes from the mice were evaluated after intranasal vaccination. We found that intranasal administration of S/O nanodispersions induced strong mucosal and systemic immune responses and could be used for the future intranasal vaccination.

[SC]PL203 Salt effect on transdermal absorption of vaccine antigen by solid-in-oil nanodispersion
Kyushu University, Fukuoka City, Fukuoka, Japan

Transdermal delivery has been developed as one of the most attractive administration methods of vaccine antigen, which is safe and convenient. In transdermal vaccination, we need to deliver antigen to the immune cell in the skin to start the immune response. However, it is difficult for hydrophilic macromolecules such as protein to be permeated into the skin due to hydrophobic barrier function of stratum corneum; the outmost layer of skin. In recent years, it has been reported that skin permeability of the antigen is enhanced by using a solid-in-oil (S/O) nanodispersion, in which hydrophilic substances were coated with a hydrophobic surfactant and dispersed in oil as nanometer-sized particles. The purpose of this research is to prepare a practical transdermal vaccine by S/O technique from commercially available injection vaccine, in which antigen is dissolved in saline because osmotic pressure of injection vaccine is adjust with body fluid. In this study, fluorescein isothiocyanate-labeled ovalbumin (FITC-OVA) and isopropyl myristate (IPM) were used as a model antigen and an oil base with skin penetration effect, respectively. To check the effects of salts on nanoparticle formation and skin permeability, FITC-OVA was initially dissolved in saline according to commercially available vaccine and prepared S/O nanodispersions. As a result, it was confirmed that the particle diameter increased and the skin penetration amount decreased in the sample of high salt concentration. It is shown that it is important to reduce the salt concentration to make a transdermal vaccine from injection one.

[SC]PL204 Design of Thermoresponsive Polymeric Surface for Continuous Culturing Human Embryonic Stem Cells
Ya-Chu LIU1, Kuan-Ju LIN2, Akon HIGUCHI1,2
1 Central University, Taoyuan, Taiwan
2 RIKEN, Saitama, Japan

Stem cells are attractive source for tissue engineering applied on regenerative medicine, translational medicine, and drug discovery. Batch type culture is the typical way for stem cell culture, which is laborious and expansive. Moreover, the digestive enzymes or EDTA solutions to detach cells are causing cell damage and increasing production costs. For reducing the costs, we developed the continuous culture system culturing the human embryonic stem cells (hESCs) on thermoresponsive polymer surfaces (Fig. 1). hESCs could be detached from the thermoresponsive polymer surfaces by reducing the medium temperature below the lower critical solution temperature (LCST) of the thermoresponsive polymer (poly-N-isopropylacrylamide and its copolymer). After reducing the temperature, the thermoresponsive surface will become more hydrophilic, creating an unfavorable environment for cell attachment. Thus, hESCs can be partially detached by gentle pipetting. The remained cells could be confluent again by adding fresh medium. This continuous culture system could be used for 3D-cultivation. However, the barrier of applying this system is hESCs became harder to attach on the thermoresponsive surfaces coating with the extracellular matrix (ECM) after multicycles. This maybe cause by degradation of ECM after long-term culture. Therefore, we investigated the precoated and uncoated methods with different ECM; recombinant vitronectin (rVN) and recombinant laminin-511 (iMatrix-511). Comparing with traditional precoated method, the uncoated method where ECM is added into culture medium upon seeding cells, provides cost-effective and time-efficient method and maintains hESCs pluripotency during long-term culture. This uncoated method applied to continuous culture system saves processing time and cost and contributes to the regenerative medicine.

[SC]PL205 Transdermal delivery of peptide in oil mediated by choline cation ionic liquid.
Kyushu University, Fukuoka City, Fukuoka, Japan

Transdermal delivery has been developed as one of the most attractive administration methods of peptides, which are used as biopharmaceuticals for refractory diseases or synthetic antigens for vaccination including cancer treatment or prevent from infections. In transdermal vaccination, we need to deliver peptide antigen to the immune cell in the skin to start the immune response. However, it is difficult for hydrophilic macromolecules such as peptides to be permeated into the skin since stratum corneum; the outmost layer of skin shows the hydrophobic barrier function. In this study, we developed oil-based formulation of peptides mediated by ionic liquid (IL) in order to achieve the efficient skin permeation. We used cytotoxic T cell epitope peptide (OVA 256-264, sequence: SIINFEKL) as a model peptide. This peptide is hydrophilic and not soluble in isopropyl myristate (IPM), which is an oil base with skin penetration effect. To disperse the peptide in IPM, the ILs consisting of choline cation and fatty acids anion were synthesized. These ILs are biocompatible and show high affinity to oil because they are derived from natural molecules and fatty acids have long alkyl chains. We successfully dispersed the hydrophilic peptide in IPM by adding the ILs and ethanol as co-solvent. Subsequently, we checked whether the peptide in oil formulation shows skin permeability and the permeation mechanism of peptide. The peptide with ionic liquid in oil formulation permeated into the model mouse skin, while the penetration of peptide in aqueous phosphate buffer was low. Moreover, we revealed that this ionic liquid in oil acts as the skin permeation enhancer by extracting and disordering the lipid in the stratum corneum. These results suggested that oil-based formulation using biocompatible ILs is a promising strategy for transdermal peptide delivery.

[SC]PL206 Evaluation of release profile and oral vaccine effects using solid-in-oil nanodispersions
Yoshiro TAHARA1, Yui ABE1, Rie WAKABAYASHI1,2, Noriho KAMIYA1,2,3, Masahiro GOTO1,2,3
1 Graduate School of Engineering, Kyushu University
2 Center for Transdermal Drug Delivery, Kyushu University
3 Center for Future Chemistry, Kyushu University

Vaccines are the most useful means for preventing infection, in which antigens are mainly administered by injection, and induce a systemic immune response in whole body to eliminate pathogens. In the case of an oral vaccine, antigens are captured M cells and delivered into Peyer's patches, lymph node-like lymphatic tissues, by the transcytosis, followed by the presentation of mucosal immunity. The ability of oral vaccines to activate mucosal immunity is attractive compared to injection because it is possible to prevent the initial infection. In this study, we tried to develop an oral vaccine delivery using a solid-in-oil (S/O) nanodispersion, in which hydrophilic drugs are coated with hydrophobic surfactants and dispersed in oil phase as nanoparticles. In addition to prevention of antigen degradation by low pH or enzymes, it is expected that the antigens are effectively delivered to the lymphatic tissues using oil-bases because hydrophobic materials such as lipids are known to be formed micelles and absorbed by lymphatic tissues rather than blood circulation. The S/O nanodispersion was prepared using ovalbumin (OVA) and sucrose fatty acid esters as a model antigen and surfactants, respectively. From in vitro release study using artificial intestinal fluid containing lipase and bile acids, the S/O nanodispersion using sucrose laurate (L-195) and perilla oil showed higher antigen release, while that using sucrose erucate (ER-290) and squalene oil released antigens gradually, resulted from the length of alkyl chain of surfactants and ester bonds of oils. From the oral vaccine study in vivo, the OVA-specific IgA production in the small intestine of mice was increased by the S/O nanodispersion using ER-290 and squalene oil compared to aqueous vehicle, phosphate buffered saline (PBS). These results suggested that the S/O nanodispersions releasing antigens gradually is suitable to deliver antigens to Peyer's patches, and induced mucosal immunity effectively.

[SC]PL207 Investigation of the effect of blood components on blood vancomycin sensor using molecularly imprinted polymer carbon paste electrode
Yuuto TAKEDA, Shunsuke HAYASHI, Akihiko HATANO, Yasuo YOSHIMI
Shibaura Institute of Technology, Tokyo, Japan

Vancomycin is the drug of first choice for treating Methicillin-Resistant Staphylococcus Aureus (MRSA) infections which is the main causative bacteria of nosocomial infection. Therapeutic drug monitoring (TDM) of VCM is strongly recommended in order to prevent side effect and creation of the resistant bacteria. However, TDM does not work sufficiently, because frequent measurement of blood level of VCM is still difficult. We developed a blood-VCM sensor with a paste electrode of graphite particle on which a molecularly imprinted polymer (MIP) is immobilized covalently in this study. The sensitivity of the sensor to the VCM concentration in whole blood and plasma was evaluated.
Experimental method.
Graphite particles were coated with photoinitiator of radical polymerization was dispersed in a polymerization solution and irradiated with xenon lamp light. The MIP carbon was mixed with silicone oil and filled in a glass tube to prepare a carbon paste electrode immobilizing MIP of VCM.
The analyte solvent was prepared by dissolving VCM in phosphate buffer, bovine whole blood or plasma, and the relationship between current obtained by differential pulse voltammetry and the VCM concentration was observed.
Results and Discussion.
The calibration line of the VCM-MIP electrode is compared in each analyte solution at Fig. 1. First, the electrode indicated sensitivity to VCM in all analyte solutions. However, the sensitivity in plasma is about twice as high as in PBS or whole blood. It can be thought he detection of VCM is not interfered by blood cells. But oxidation of uric acid, or other kind of oxidative species in plasma enhances background current and oxidative current of vancomycin. But the enhancement is probably suppressed by the blood cells. However, the result indicates that the VCM-MIP electrode enables the sensing of VCM in whole blood without separation between the plasma and blood cells. The property is advantageous for real time monitoring of VCM.

[SC]PL208 Comparative study in different motifs of laminin-derived synthetic peptides for adhesion, proliferation and differentiation of human pluripotent stem cells
Ming-Wei LU1, Huan-Chiao SU1, Akon HIGUCHI1,2
1 National Central University, Taoyuan, Taiwan
2 RIKEN, Wako, Japan

Human pluripotent stem cells (hPSCs) includes human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are a promising source for regenerative medicine and tissue engineering. Typically, hPSCs are used to culture on the extracellular matrix (ECM) coated dishes, such as Matrigel, recombinant vitronectin (rVN) and laminin-521 (LN-521), which were secreted from animal or human cells. However, those ECMs provided uncertain chemical composition. Therefore, the xeno-free synthetic biomaterials offer not only a chemical defined composition but also a reproducible culture condition.
In our previous study, we successfully cultured hPSCs on polyvinylalchohol-co-itaconic acid (PVA-IA) hydrogels, with optimal crosslinking elasticity, conjugated with vitronectin-derived oligopeptides (KGGPQVTRGDVFTMP), which provide RGD binding motifs for cell adhesion. The further key cell binding mechanism will be investigated by using PVA-IA hydrogels grafted with synthetic peptides from different chain compositions. Those synthetic peptides chains contain five variants of α-chain, four variants of β-chain and three variants of γ-chain of laminin, respectively. These synthetic peptides will be investigated by comparing the ability to support long term hPSCs culture and differentiation into cardiomyocytes (CMs). Therefore, we prepared PVA-IA hydrogels grafted with different oligopeptide sequences derived from Laminin α-chain (RGD), α1-chain (IKVAV) and β1-chain (YIGSR and PDSGR) to compare the efficiency of cell attachment and differentiate into CMs (Fig. 1). We also design some motifs with different length of joint segment to mimic the original ECM and evaluate the length effect on the culture of hPSCs. In this study, the expression four pluripotency gene (Oct3/4, Klf4, Sox2, and Nanog) of hPSCs is evaluated for ability to support hPSCs growth. hPSCs are induced into cardiomyocytes to screen the optimal synthetic peptide. We expected to screen the optimal combination of synthetic peptides for hPSC culturing and differentiation.

[SC]PL209 Transcutaneous delivery of peptide antigen with reverse micelles for a development of cancer vaccine
Shuto KOZAKA1, Takahiro NAKATA2, Shiro TAJIMA2, Taro UEDA2, Masahiro GOTO1
1 Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka, Japan
2 Kobayashi Pharmaceutical Co., Ltd. 1-30-3 Toyokawa, Ibaraki, Osaka, Japan

Cancer vaccine, a prophylactic therapy against cancer by administering a cancer antigen to activate the host immunity, has come to draw a great deal of attention due to its low risks of side effects. In this study, we focused on the transcutaneous drug delivery system for the cancer antigen delivery. This delivery route would be the ideal way, because skin immune cells are known to induce a strong immune response. However, the stratum corneum (SC), the outermost layer of the skin, works as a hydrophobic barrier to prevent the penetration of antigens which are mostly hydrophilic. To overcome this problem, we tried to use Reverse Micelle Formulation (RMF). In RMF system, hydrophilic drugs are enclosed inside the aqueous core of reverse micelles and dispersed in oil phase which SC prefers. Despite there were numerous reports regarding transcutaneous delivery using RMF, there was no report on a transcutaneous vaccine due to its still low skin permeability. In this study, we used amphiphilic Skin Permeation Enhancer (SPE) as surfactant which forms reverse micelles to realize even higher permeability. To the best of our knowledge this is the first report on this strategy, and we conducted experiments to prove its effectiveness.
We chose K-TRP-2 (KKKGSVYDFFVWL) peptide as a cancer antigen, and fatty acid glycerol ester as amphiphilic SPE. By dropping the aqueous solution of antigen onto surfactant-containing oil, we obtained uniform RMF. As a result of in vitro experiment, RMF showed a significant improvement in skin permeation of antigens. Also, RMF enabled the successful antigen delivery to the skin immune cells, which is a key step to induce an immune response, and its transcutaneous treatment recruited more cytotoxic T lymphocytes into tumor microenvironment than an injection treatment and strongly suppressed a tumor growth.

[SC]PL210 Preparation of microporous hydrogel sponges for 3D perfusion culture of mammalian cells
Aruto HORI, Rie UTOH, Masumi YAMADA, Minoru SEKI
Chiba University, Chiba City, Chiba, Japan

Preparation of three-dimensional (3D) organ models composed of liver cells is expected as an efficient approach to in vitro cell-based drug screening and development. Various types of hydrogel-based 3D cell culture systems have been developed, but the lack in the capillary networks in hydrogel matrices causes severe problems in terms of insufficient supply of oxygen and nutrition. Here we proposed a facile strategy for creating perfusable hydrogel-based liver cell culture systems. We utilized a bicontinuous aqueous two-phase dispersion, which was composed of polyethylene glycol (PEG)-rich and gelatin methacrylate (GelMA)-rich phases, to produce cell-encapsulating microporous gelatin-based hydrogels. The interconnected pores were utilized to perform perfusion culture of encapsulated HepG2 cells.
We first prepared an aqueous two-phase system of PEG/GelMA, and then remixed the separated two phases by controlling the mixing ratio, with the addition of a photopolymerization initiator and cells. Immediately after mixing, the GelMA-rich phase was photocrosslinked by irradiating UV light, to form millimeter-sized tablets of microporous sponges. We confirmed that most of the encapsulated cells were viable during this mixing and crosslinking processes, and after cultivation for 1 week. In contrast, cells encapsulated in uniform GelMA hydrogel were mostly dead after 1 day of cultivation. The expressions of hepatocyte-specific genes were upregulated for the hydrogel sponges, which was confirmed by RT-qPCR. Additionally, we performed perfusion culture of the cells, by packing the obtained hydrogel sponges into microfluidic perfusion chambers, and evaluated the functions of the encapsulated cells. The presented hydrogel sponges are useful because we did not need any complicated devices or protocols for preparing hydrogel sponges, and they would be applicable as a unit platform for organs-on-a-chip systems.

Minh Phuong NGUYEN, Dukjoon KIM
Sungkyunkwan University, Suwon City, Gyeonggido, South Korea

In this study, we report synthesis of hydropholic surface and active-target superparamagnetic iron oxide nanoparticles (SPIONs) for hyperthermia cancer therapy. Superparamagnetic iron oxide nanoparticles (SPIONs) capping oleic acid were prepared by thermal decomposition method. The synthesized iron oxide nanoparticles were encapsulated with the polyaspartamide (PA) to enhance their biocompatibility and hydrophilicity (PA-encapsulated SPIONs). A polysuccinime (PSI) which is biocompatible and biodegradable is the backbone of PA. In addition, multifunctional polymers including hydrophilic O-(2-aminoethyl) polyethylene glycol (PEG) and hydrophobic octadecylamine (C18) were grafted on PSI for amphiphilic structure, and on biotin for increasing the cancer cellular uptake. The diameter of the PA-encapsulated SPIONs was confirmed by TEM and DLS. The structure of the polyaspartamide was confirmed by 1H NMR. The structure and biomedical properties of PA-encapsulated SPIONs were investigated in vitro and in vivo experiments.

[SC]PL212 Development and immunological evaluation using solid-in-oil-in-water adjuvant
Riko MIZUNO1, Yoshiro TAHARA1, Rie WAKABAYASHI1,2, Noriho KAMIYA1,2,3, Masahiro GOTO1,2,3
1 Graduate School of Engineering, Kyushu University
2 Center for Transdermal Drug Delivery, Kyushu University
3 Center for Future Chemistry, Kyushu University

A vaccine is a drug that administers an antigen derived from a pathogen to the body and induces antigen-specific immunity. In the case of therapeutic vaccines, it consists of antigens and adjuvants (additives to enhance the immune response), and various delivery systems that deliver them to immune cells have been reported. In several studies, strong vaccine effects were induced using intelligent lipid or polymer-based delivery systems. However, most of the candidates have not been put to practical use because novel, synthesized compounds must be confirmed the safety in human. Mostly, it takes over 10 years to accept to use clinical phase, and it is hard to balance high safety and effect of vaccine delivery. To overcome this problem, we used a clinically accepted adjuvant, oil-in-water (O/W) emulsion, as vaccine carrier. Though it has high safety and effect for human, the efficiency is limited because hydrophilic antigen and O/W emulsion are administered separately. In this study, we tried to create a solid-in-oil-in-water (S/O/W) adjuvant that encapsulates antigens in oil phase using solid-in-oil (S/O) technology and can simultaneously administer antigen and adjuvant. Composition of S/O/W adjuvant is based on MF59, commercially available O/W emulsion, and ovalbumin (OVA) was used as a model antigen. The S/O/W adjuvant was determined as a nano-sized emulsion (ca. 300 nm) and over 90 % of antigen was encapsulated after 24 h. From in vitro study using dendritic cells, efficient intracellular delivery and lysosomal escape of antigens were achieved by S/O/W adjuvant. Furthermore, when S/O/W adjuvant was administered subcutaneously to mice in vivo, the antigen-specific humoral and cellular immunity was induced more effectively than O/W emulsion and OVA free. These results suggested that S/O/W adjuvant, which can achieve co-delivery of antigen and adjuvant, is a novel approach to develop effective carriers for therapeutic vaccine such as cancer immunotherapy.

[SC]PL213 Production of influenza virus proteins using recombinant insect cells
Kobe University, Kobe, Japan

Virus-like particles (VLPs) can be produced using recombinant DNA technology by expressing viral surface proteins that spontaneously assemble into particulate structures similar to authentic viral particles. VLPs serve as safe and effective vaccines and diagnostic antigens. The baculovirus–insect cell system has been used extensively for the production of a wide range pf VLPs. However, the baculovirus system has several inherent limitations including contamination of VLPs with progeny baculoviruses. Stably transformed insect cells can be used as attractive alternatives to the baculovirus–insect cell system. In the present study, the production of influenza VLPs by recombinant insect cells was investigated.
The DNA fragments encoding hemagglutinin (HA) and matrix protein 1 (M1) of an influenza virus A (H1N1) were individually cloned into the plasmid vector pIHAbla and pIHAneo. The pIHAbla and pIHAneo contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression, together with either a blasticidin or a neomycin resistance gene for use as a selectable marker, respectively (Yamaji et al.: Biochem. Eng. J., 41, 203–209 (2008)). After cotransfection with the resultant plasmids, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were incubated with blasticidin and G418, and cells resistant to the antibiotics were isolated.
Western blot analysis of a culture supernatant showed that transfected High Five cells secreted HA and M1. Sucrose density-gradient sedimentation analysis and dynamic light scattering of the culture supernatant suggested that secreted HA and M1 molecules were produced in a particulate form. Hemagglutination assay using chicken erythrocytes revealed hemagglutination activity in the culture supernatant. Taken together, recombinant insect cells may offer a promising approach to the development and production of influenza VLPs.

[SC]PL214 pH-triggered intracellular release of doxorubicin from polyaspartamide-encapsulated mesoporous silica nanoparticles
Cheolwon LIM, Dukjoon KIM
Sungkyunkwan University, Suwon, Gyeonggi, Korea

Mesoporous silica nanoparticles (MSNs) conjugating doxorubicin (DOX) via a pH-sensitive cleavable linkage, hydrazine (HYD) were synthesized. MSN-HYD-DOX were encapsulated with the polyaspartamide (PASPAM) grafted with the hydrophilic o-(2-aminoethyl)-o′-methylpoly(ethylene glycol) (PEG) and the cell permeating ligand, biotin (Biotin). The chemical structure of the synthesized MSN-HYD-DOX and PASPAM-g-PEG/Biotin was confirmed using FT-IR and 1H-NMR spectroscopy. The mean diameter of the MSN-HYD-DOX@PASPAM-g-PEG/Biotin nanoparticle was 142 nm and 121 nm, respectively, examined by dynamic light scattering (DLS) and transmission electron microscope (TEM). The HYD bond was effectively cleaved in acidic condition, and thus DOX was released much faster at pH 5.0 than at pH 7.4. The cell viability in MSN-HYD-DOX@PASPAM-g-PEG/Biotin system was much lower than that of the free DOX drug because of efficient intracellular drug delivery associated with the biotin ligand.

[SC]PL215 Retrotransposon-mediated gene transfer of scFv-Fc gene into Chinese hamster ovary cells
Feiyang ZHENG1, Yoshinori KAWABE2, Mai MURAKAMI1, Mamika TAKAHASHI2, Shoichiro YOSHIDA2, Akira ITO2, Masamichi KAMIHIRA1,2
1 Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, Japan
2 Department of Chemical Engineering, Faculty of Engineering, Fukuoka, Kyushu University, Japan

Gene delivery methods for animal cells are one of the most important tools in biotechnology fields such as gene therapy, generation of transgenic animals and pharmaceutical protein production. We have attempted to develop a gene transfer method based on retrotransposon. Retrotransposons are mobile elements that transfer themselves in a cell genome by the 窶彡opy-and-paste窶・mechanism. The process is known as retrotransposition. A retrotransposon, long interspersed element-1 (LINE-1, L1) is categorized as a non-LTR retrotransposon, comprised of a 5' untranslated region (5'UTR), two non-overlapping open reading frames (ORFs) separated by a short inter-ORF sequence, and a 3'UTR that ends in an adenosine-rich tract. We constructed an L1-based vector plasmid including the essential components for retrotransposition. An intron-disrupted Neor reporter gene and a scFv-Fc expression unit under the control of CMV promoter were inserted into 3'UTR region in order to evaluate retrotransposition and scFv-Fc production. CHO-K1 cells transfected with the plasmids were screened using G418 for 10 days. The established cell clones produced scFv-Fc proteins in the culture medium. To control retrotransposition, we removed the ORF2 sequence from L1 vector to generate ORF2-deleted L1 (L1[$Delta;orf2]) vector, and constructed ORF2 expression vectors (pORF2 and pORF1-2) for providing ORF2 proteins. Under the optimal ratio of L1[$Delta;orf2] vector and ORF2 expression vector, no significant difference in the number of G418-resistant colonies formed was observed compared with that for L1 vector although it slightly decreased. The scFv-Fc gene was detected in all clones by genomic PCR. Real-time PCR revealed that scFv-Fc gene was integrated into the genome at the range from one to six copies in the clones.

[SC]PL216 Design of biomaterials immobilized ECM and ECM-derived peptides for pluripotent stem cells differentiation
Tzu-Cheng SUNG1, Hsing-Fen LI1, Cheng-Hui LIU1, Akon HIGUICHI1,2
1 Department of Chemical and Materials Engineering, Central University, No. 300, Jhongda RD., Jhongli, Taoyuan 32001, Taiwan
2 Nano Medical Engineering Laboratory, RIKEN, Wako, Saitama351-0198, Japan

Stem cells are an attractive prospect for regenerative medicine and tissue engineering. However, stem cell are difficult to differentiate into specific cell types. Thus, reliable methods to differentiate stem cells into desired cell types are necessary to be developed. Here, we developed nanosegment-grafted biomaterials having different elasticity. [1] We also developed optimal differentiation media to induce the differentiation of hES cells into cardiomyocytes. We developed several biomaterials having different elasticity for hPSCs differentia-tion into cardiomyocytes. We prepared (1) ECM (extracellular matrix)-coated dishes, (2) PVA-IA (polyvinylalcohol-co-itaconic acid) hydrogel dishes having different elasticity that are grafted with several ECMs [2], and (3) PVA-IA hydrogel dishes having different elasticity that are grafted with cell-adhesion oligopeptide (oligovitronectin). We also investigated the differentiation efficiency using different induction medium: (a) Commercial cardiomyocyte induction medium, (b) RPMI 1640 medium supplemented with B27 and bovine serum albumin (BSA), (c) CDM3 medium (xeno-free culture medium) developed by literature [3] and in this study. On day 0, we replaced the expansion medium into cardiomyocytes differentiation medium containing the GSK3β inhibitor. On days 1-2, we observed that 30%~40% of the cells in the medium shown in medium (a) and 50~60% of the cells in the medium (b) were died and detached from the surface. However, the center of the colony of living cells were getting thicker and became compact. These cells were differentiated into cardiomyocytes on days 5-6. On day 8-10, we successively observed the contracting colonies on each biomaterial surface. We successfully screened the optimal elasticity of bio-materials, preferable nanosegments immobilized on the biomaterials as well as the cell culture medium for the optimal differentiation of hPSCs into cardiomyocytes. This system will be used for developing for cell sorting system, which will be a great benefit to its clinical application in regenerative medicine.

[SC]PL217 Generation of gene-engineered human hepatoma cells with heat-inducible liver functions
Hiroyuki KITANO1, Manuel SOUVERVIELLE2, Yuto SONODA1, Yoshinori KAWABE1, Akira ITO1, Masamichi KAMIHIRA1
1 Dept. Chem. Eng., Fac. Eng., Kyushu Univ., Fukuoka, Japan
2 Grad. Sch. Syst. Life Sci., Kyushu Univ., Fukuoka, Japan

For acute and fulminant liver failure patients, liver transplantation is only an effective treatment. However, it has a big problem in donor organ shortage. As a bridge use for liver transplantation, bio-artificial liver (BAL) systems with extracorporeal blood circulation have been an attractive choice. For constructing BAL systems in which functional hepatic cells are filled in a bioreactor, hepatoma cells derived from liver carcinoma are a candidate cell source due to their high proliferative capacity, although liver function of hepatoma cells is considerably lower than that of primary hepatocytes. In our previous study, genetically engineered mouse hepatoma cells with inducible high liver function were established by transducing liver-enriched transcription factor (LETF) genes (Yamamoto et al., Biochem. Eng. J., 2012, 60, 67-73; J. Biosci. Bioeng., 2018, 125, 131-139). In this study, human hepatoma HepG2 cells were genetically engineered to express liver function in response to heat treatment. For this purpose, we constructed a heat-inducible expression of LETF genes, in which a tetracyclin-responsive artificial transactivator is expressed under control of a heat-shock protein promoter with transcriptional amplification and the transactivator then induces LETF genes. The gene expression system was constructed on transposon vectors and introduced into HepG2 cells. Transgenic cells (HepG2/HSP/8F) were screened by drug (puromycin and blasticidin) selection. For inducing overexpression of LETF genes, cells were heated at 43 °C for 30 min using water bath. Albumin secretion and cytochrome P450 activity were measured for the cells with or without heat treatment. The liver functions of HepG2/HSP/8F cells were enhanced by the heat treatment. The cells with heat-inducible liver function can be a new cell source for various hepatic studies including construction of BAL systems.

[SC]PL218 One-step encapsulation of water-insoluble bioactive compounds into chitosan-oleic acid complex particles and their characteristics as food dispersions
Hiroki SHINO, Tatsuhito SUZUKI, Azumi IIDA, Akihiko KANAZAWA, Takashi KUROIWA
Tokyo City University, Tokyo, Japan

Encapsulation of water-insoluble bioactive compounds using chitosan-oleic acid complex particle was investigated. One-step preparation of chitosan-oleic acid complex particles encapsulating bioactive compounds with a narrow diameter distribution was carried out by the simple mixing of a chitosan aqueous solution and an ethanol solution containing oleic acid at room temperature. The formation of particles with mean diameter below 1 micrometer was confirmed by dynamic light scattering and laser diffraction measurements. A characteristic peak indicating aggregation of alkyl chain of oleic acid molecules was observed by small angle X-ray scattering measurement, suggesting that the particle had hydrophobic domains formed by aggregation of oleic acid molecules inside the complex particles. These characteristics agreed with scanning-transmittance electron microscopic images of complex particles. Various water-insoluble compounds including curcumin, capsaicin, and vitamin E were successfully encapsulated into the complex particles. Diameter distribution of the complex particles encapsulating bioactive compounds were maintained during storage over a month, suggesting that the particles were highly stable. The particle suspension could be concentrated by evaporation and powderized by freeze-drying without significant changes in their diameter. The results demonstrated that our method for encapsulating water-insoluble hydrophobic bioactive compounds using chitosan-oleic acid complex particles were potentially applicable as edible materials for development novel food dispersion systems.

[SC]PL219 Evaluation of pharmacokinetics using the different base in local anesthesia in vitro
Ko OHIRA1, Yoshiro TAHARA1, Koji HARADA2, Masahiro GOTO1,3,4
1 Graduate School of Engineering, Kyushu University
2 Graduate School of Medicine, Yamaguchi University
3 Center for Transdermal Drug Delivery, Kyushu University
4 Center for Future Chemistry, Kyushu University

A local anesthesia (LA) relieves the sense of a specific part of the body. It is used in the medical procedure for needle puncture or venous cannulation. The main administration of LA is injection, but this method is not only invasive and painful but also requires specialized knowledge and risk of secondary infection, so an alternative administration is required. Transdermal delivery of LA has advantages that enable to bring anesthesia effect without any pain and difficulty. The transdermal LA is available in commercial, but the current marketed product is not popular because it cannot eliminate pain completely and needs long-term continuous administration. Therefore, we aim to improve the effect of transdermal local anesthesia using non-aqueous base materials. Previously, the relationship of the pharmacokinetics and pain-relieving effect of LA are not revealed completely. These facts make difficult to improve analgesic effectiveness of LA. In this study, we conducted early clinical study in human and compared the results obtained in clinical trials with in vitro studies in laboratory scale. We used lidocaine as LA, and polyethylene glycol 400 (PEG400), isopropyl myristate (IPM) and isopropyl alcohol (IPA) as the base. In clinical study, we found that LA using IPA induced actual analgesic effectiveness and achieved painlessness within 10 minutes. On the other side, we found that IPM showed high permeation across the pig skin in vitro. It is suggested that high permeability across the skin did not lead improving analgesic effectiveness. Subsequently, we measured the amount of drug in the skin, and the sample using IPA as the base showed highest amount of lidocaine in the skin (shown in Figure 1, SC: stratum corneum). It is suggested that staying drug in the skin leads improving actual analgesic effectiveness because the result of clinical study corresponds with the result in vitro.

[SC]PL220 Transdermal delivery of cedar pollen epitope peptide by a solid-in-oil nanodispersion.
Kyushu University, Fukuoka, Japan

In recent years, the number of Japanese cedar pollinosis patients are increasing, and pollinosis immunotherapy has attracted much attention. However, the administration of a whole pollen allergen by injection has the risk of side effects such as anaphylactic shock. In this study, we tried to develop a simple, non-invasive method of pollinosis immunotherapy by the way of transcutaneous administration and used cedar pollen T cell epitope peptide A (PepA: SMKVTVAFNQFGP) with a low risk of adverse effects as an antigen. The presence of stratum corneum (SC) which is the hydrophobic barrier in the outermost layer of the skin makes it difficult for the hydrophilic antigen to penetrate the skin. To overcome the barrier, antigens were dispersed in hydrophobic solvent as nano-order particles using the solid-in-oil (S/O) nanodispersion, in which antigens are coated with hydrophobic surfactant molecules, to permeate through the SC. In this study, K-PepA, in which hydrophilic trilysin was conjugated to PepA, was used to increase the solubility in water and stability of the S/O nanodispersion.
First, the mice were vaccinated with K-PepA thrice with a one-week by injection. One week after the final immunization, splenocytes were harvested and T cells were quantified using flow cytometry. Similar to PepA, an increase in the number of Th1 cells was observed by K-PepA, suggesting the efficacy for pollinosis immunotherapy. Second, the S/O nanodispersion was obtained by freeze-drying the water-in-oil emulsions containing K-PepA in water phase and surfactant, a sucrose laurate in oil phase, and dispersing it in isopropyl myristate. The permeability of K-PepA enclosed in S/O nanodispersion to mouse skin was evaluated in vitro and a significant improvement in permeability was confirmed as compared with the PBS solution. These results suggest that the S/O nanodispersion containing K-PepA is a promising approach to effective transcutaneous pollinosis immunotherapy.

[SC]PL221 Transdermal peptide delivery using ionic liquid consisting of amino acid as cation
Kyushu University, Fukuoka, Japan

Transdermal drug delivery system (TDDS) is a drug administration method that has attracted attention in terms of safety, convenience, and low invasiveness. However, it is difficult for hydrophilic micromolecular drugs to be delivered into the skin surface since the stratum corneum, which is the outermost layer of the skin, has a hydrophobic barrier function. Therefore, it is effective to use a method of dispersing drugs in an oil base having a transdermal penetration promoting effect, but hydrophilic drugs are not soluble in oil base. In this study, the use of ionic liquids (ILs) to solubilize poorly soluble substances was considered. It is important that ILs are low toxicity and consist of biocompatible materials. So far, choline has been used as a cation in many reports on the application of ILs to TDDS. In this presentation, we synthesized a new biomaterial-based IL using ethyl esterified β-alanine as the cation, and compared with a choline-based IL. In both ILs, oleic acid was used as the anion, which is expected to have high affinity to oil base. We selected ovalbumin epitope peptide as a model drug. The peptide is hydrophilic and was insoluble in oil base, isopropyl myristate (IPM), but the addition of the β-alanine-based IL succeeded in dispersing the peptide in IPM. On the other hand, the peptide is not clearly dispersed in a simple mixture of IPM and choline-based IL. Furthermore, when the permeability of the peptide to pig skin in vitro was evaluated, it was hardly penetrated by the aqueous phosphate buffer solution because of stratum corneum's hydrophobic barrier function, but showed high permeability by the solution dispersed in IPM using β-alanine-based IL. These results suggest that the combination of a new biomaterial-based IL using amino acid as the cation and IPM is a useful strategy for TDDS.

[SC]PL222 Improvement of immunity in transcutaneous vaccine by a combination of skin permeation enhancer and adjuvant
Junpei KANEKO1, Yoshiro TAHARA1, Momoko KITAOKA1, Rie WAKABAYASHI1, Noriho KAMIYA1,2, Masahiro GOTO1,2
1 Department of Applied Chemistry, Graduate School of Engineering, Kyushu University
2 Center for Future Chemistry, Kyushu University

Vaccine is the most effective way to prevent infection by administrating antigen to enhance immunity. Currently, most vaccines are administrated by subcutaneous injection, but it has some problems. Therefore, transcutaneous immunization has attracted attention as a new vaccination method. Transcutaneous immunization is a novel vaccination strategy that delivers antigens to intact skin topically to induce protective immune responses. This needle-free strategy is easy-to-use, safe, and low invasive compared with conventional subcutaneous injection. The starting point of transcutaneous immune mechanism is a capture of antigen by dendritic cells (DCs) in the skin. DCs as a strong immune response inducer, could be expected by the transcutaneous route. However, the skin surface is covered with hydrophobic stratum corneum, which make it difficult to deliver hydrophilic antigens to DCs. To overcome this difficulty, we developed a novel formulation of nano-dispersed solid-in-oil (S/O). The novel S/O formulation can be prepared only by high-speed stirring, and it can contain various additives in large amounts. In this study, we tried to improve the vaccine effect by two approaches of promoting skin permeation and activating immune cells, by adding skin permeation enhancer and adjuvants to the novel S/O formulation.
On the study of antigen uptake by DCs, after 12 hours of vaccine administration to mice, skin cells were analyzed by flow cytometer. As a result, the novel S/O formulation containing skin permeation enhancer and adjuvant significantly increased the antigen delivery rate to the skin DCs, it is 4 times of aqueous solution. In the evaluation of vaccine efficacy, blood antibody level after vaccine administration was measured. As a result, the novel S/O formulation induced antibody production equivalent to injection.
In conclusion, by adding skin permeation enhancer and vaccine adjuvants to the novel S/O formulation, we succeeded the development of transcutaneous vaccine which has an effect comparable to injection.

[SC]PL223 Enhancement of transdermal delivery of biomolecules by reverse micelles
Ayaka KASHIMA1, Shuto KOZAKA1, Takahiro NAKATA4, Shiro TAJIMA4, Taro UEDA4, Masahiro GOTO1,2,3
1 Department of Applied Chemistry, Graduate School of Engineering, Kyushu University
2 Advanced Transdermal Drug Delivery Center, Kyushu University
3 Center for Future Chemistry, Kyushu University
4 Kobayashi Pharmaceutical Co., Ltd.

In recent years, biopharmaceuticals have attracted much attention. This is because they are considered effective for diseases that cannot be treated with conventional small drug molecules. Therefore, there is a need to establish a useful method for administering biopharmaceuticals to patients. At present, administration methods for such biopharmaceutical are limited to injection, because oral administration degrades protein molecules, which are the main active ingredients of pharmaceuticals, by digestive enzymes. On the other hand, injection administration is less convenient because it requires a medical doctor, and it often involves the risk of needle stabbing and pain. In the present study, we aim to establish a simple and safe administration method to replace injection and focus on transdermal administration. For the purpose, we need to overcome the serious problem of transdermal administration, which is a strong barrier of skin surface for transdermal delivery of biomolecules. In this research, we proposed a novel drug carrier called 窶徨everse micelles窶・for the transdermal drug delivery. Reverse micelles are oil-based molecular assembly, in which various kinds of biomolecules can be entrapped into the water cores. Focusing on the fact that the skin surface is hydrophobic, we thought that the entrapped biomolecules in reverse micelles could permeate into the skin. This reverse micellar method has been used for small molecule drugs so far, however it has not been applied to large biomolecules such as peptides or proteins. In this study, we have developed reverse micelle formulation that enables successful transdermal delivery of biomolecules, and discussed the permeation mechanism by reverse micelles.

[SC]PL224 Development of xenoantigen-detection tools for manufacturing pharmaceutical protein and cell-processing material for reproductive medicine
Noriyuki YUASA, Kento KAWAMURA, Takahiro TANJI, Yoshihide NISHIKAWA, Takashi OTA, Jun IWAKI, Hideki ISHIDA, Yuji MATSUZAKI
Department of Glycotechnology, Tokyo Chemical Industry Co., Ltd

In the production of pharmaceuticals, xenoantigens derived from animal cells and animal-derived materials may be present in the final products. These compounds are often problematic and must be identified. Among them, xenoantigen carbohydrates have immunogenic effects and difficult to remove from drugs because they bind to proteins.
Anti-αGal is a natural antibody in humans. Binding of this antibody to αGal antigens (αGal epitope) expressed on porcine xenograft surfaces is a major factor affecting engraft survival. Recently, studies showed that some therapeutic antibodies and cell processing materials used in reproductive medicine contain the αGal epitope. Another major xenoantigen is N-glycolylneuraminic acid (Neu5Gc). An enzyme is responsible for Neu5Gc synthesis; when the gene encoding this enzyme is mutated, humans cannot synthesize Neu5Gc. Humans produce antibodies against Neu5Gc glycan as heterogenous structures, causing the immunogenicity of therapeutic proteins containing Neu5Gc glycan epitopes. Therefore, rapid and robust methods for detecting the αGal epitope and Neu5Gc are required. We chemically synthesized various structure-defined N-glycans including human-type glycans (G0, G2, and SG), uniform isomers (6-G1 and 3-G1) and heterogenic antigens including αGal or Neu5Gc. We also produced high-grade 2-AB-labeled N-glycans. These compounds are suitable for mass spectrometry, capillary electrophoresis, and high-performance liquid chromatography analyses. We simultaneously developed polyclonal antibodies that specifically bind to xenoantigen carbohydrate, αGal, and Neu5Gc using chicken. Some antibodies were modified with fluorescent probes or enzymes for versatile detection.
In this poster, we introduce new methods for detecting xenoantigen carbohydrates in cell culture supernatants or purified antibodies with our reagents. Xenoantigen carbohydrate attached to Fc and/or Fab regions of the antibody can be detected using our methods. We also introduce a method for detecting xenoantigens in the cell culture supernatant in the early stages of cell establishment.

[SC]PL225 Control of drug self-assembly behaviors by designed pH-responsible pro-drug molecules
Jin HAN1, Keishi SUGA1, Keita HAYASHI2, Yukihiro OKAMOTO1, Hiroshi UMAKOSHI1
1 Osaka University, Toyonaka, 1-3 Machikaneyama-cho
2 National Institute of Technology, Nara College, Yamato-Koriyama

Biological membranes are dynamic and heterogeneous lipid bilayer structures involved in numerous cellular processes, not only as simple boundary but also active uptake of external molecules. A new approach, termed as membrane-lipid therapy, has proved the possibility that some of lipophilic molecules can show therapeutic effect as like drug, by interacting with biomembrane. Therefore, the alteration of lipid membrane properties has been expected to promise a novel and great potential of therapeutic effect. Here, a self-assembled pro-drug system have been investigated, focusing on controlling the self-assembly behavior by surroundings such as pH. Pro-drug structures were inspired from self-assembling fatty acids (FAs). FAs are amphiphilic molecule with hydrophobic carbon chain and hydrophilic carboxyl group, whose phase states strongly depend on pH condition and molecular structure. The aryl carboxylic acids Oxaprozin (Oxa), a main kind of nonsteroidal anti-inflammatory agent, was chosen as model molecule and designed to Oxa-lipid prodrug molecules possessing carboxyl group with different carbon chain length and achieved control of drug self-assembly behavior under different pH conditions. Designed pro-drug molecules achieved the control of drug self-assembly behaviors based on pH condition, which could realize the development of biological microenvironment-sensitive drug delivery system. It can be expected that the variation of self-assembly structure might contribute to enhanced therapeutic efficiency.
Reference: P.V. Escriba et al., Trends Mol. Med., 12, 34 (2016). Han J. et al., J. Phys. Chem. B, 121, 4091 (2017). M. Bucciantini et al., FASEB J., 26, 818 (2012).

[SC]PL226 Designer drug delivery carriers constructed through a complementary interaction introduced to self-assembling peptide amphiphiles and a small molecular drug
Hiroki OBAYASHI1, Rie WAKABAYASHI1, Noriho KAMIYA1,2, Masahiro GOTO1,2
1 Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Japan
2 Division of Biotechnology, Center for Future Chemistry, Kyushu University, Japan

Supramolecular polymers based on molecular self-assembly has attracted attention in fabricating bio-functional materials because of design diversity and biomimeticity in structural and functional aspects.[1] Self-assembling peptide amphiphiles (PAs) undergo spontaneous supramolecular assembly in aqueous solutions and have shown a potential in the use in biomedical applications, such as drug delivery carriers. In this study, we developed a novel co-assembly system based on PAs to fabricate a carrier that enables the control of assembled structures. To encapsulate small molecular drugs into PA nanomaterials, we introduced a complementary hydrogen bonding interaction into PAs and drug derivatives. Cyanuric acid (Cya) and melamine (Mel) derivatives are known complementary hydrogen bonding pair, which forms 3:3 network structure called rosette, thus we synthesized Cya-conjugated PAs (Cya-PAs) and Mel-attached model drug (Mel-NBD). To vary the assembled structures to study the influence of the structural aspects on the efficiency in intracellular drug delivery, several hydrophobic substituents of Cya-PAs were introduced; from C0 (Cya-PA1), C6 (Cya-PA2), to C12 (Cya-PA3). When Cya-PAs and Mel-NBD were mixed in a 1:1 molar ratio, they formed distinct nano-assemblies. Interestingly, the morphologies and dimensions of the assemblies were highly dependent on the PA design; i.e. globular objects (~100 nm) were observed with Cya-PA1, whereas fibrous assemblies were obtained with Cya-PA2 (2-300 nm) and Cya-PA3 (> 1 μm). In vitro study on intracellular delivery of Mel-NBD using HeLa cells showed that the cellular internalization efficiency increased in the form of assembly compared with Mel-NBD alone. Moreover, the cellular uptake was differed by the size of the carrier. These results indicate that this co-assembly system provides controllable nanostructures for an efficient delivery of small molecular drugs.
[1] R. Wakabayashi et al., Chem. Commun., 2019, 55, 640–643.

[SC]PL227 Neuronal differentiation of NT2 cells in monolayer and spheroid cultures
The University of Kitakyushu, Kitakyushu, Fukuoka, Japan

Metabolism and differentiation of culture cells are influenced by changes in cellular morphology, cell-cell and/or cell matrix interactions, and medium components, and so on. To control the destiny of cells, it is important to understand the relationship between these parameters and cell properties. In this study, we focused on the effects of cellular morphology on cell properties. Monolayer culture has been widely used as a conventional culture technique. On the other hand, a spheroid (spherical organoid) culture has been established as typical three-dimensional culture technique. In this study, we investigated the difference of cell proliferation and neuronal differentiation of NT2 cells, model cell of neural research, in the monolayer and spheroid cultures.
For the spheroid culture, we fabricated the microwell chip which consisted of 195 circular microwells (600 ホシm in diameter) on a poly-methylmethacrylate plate, and the surface was modified with polyethylene glycol to promote spheroid formation. Traditional tissue culture plate was used for monolayer culture. NT2 cells were cultured in a medium containing retinoic acid (RA), an inducer of neural differentiation.
In the monolayer culture, the cells adhered and extended on the plate. In the spheroid culture, the cells were aggregated in each microwell and formed the spheroid within 24 hours of culture, and the spheroid morphology was maintained during culture period. Although RA treatment suppressed the cell proliferation in both culture systems compared to RA non-treatment condition, the cell proliferation ability in monolayer culture was higher than that in spheroid culture. The neuronal differentiation ability of NT2 spheroid culture was higher than that of monolayer culture, and the differentiation ability was dramatically enhanced by RA treatment.
These results indicate that the proliferation and differentiation properties of NT2 cells are influenced by the difference of cell morphology.

[SC]PL228 Development of histidine-containing amphiphilic peptide as a carrier for paclitaxel: Stimuli-responsive dispersibility by adding divalent metal ions
Miyazaki University, Miyazaki, Japan

Stimuli-responsive drug delivery systems have attracted attention because of their potential to maximize therapeutic effects and minimize side-effects. Recently, authors have developed an amphiphilic short-chain peptide (KHHA7), which consists of a hydrophobic domain based on seven alanine residues and a hydrophilic domain based on a lysine and two histidine residues (KHH), as a dispersant for a poorly water-soluble anticancer drug paclitaxel (Ptx). As the histidine residues form a stable chelate complex with intermediate metal ions such as Cu(II) and Ni(II), the dispersibility of the complex between Ptx and KHHA7 alters in the presence or absence of metal ions. In the present study, two analogs of KHHA7 (KHHA3WA3 and KHHA6W), in which one of the alanine residues in KHHA7 was substituted to a tryptophan residue, were prepared to investigate the effect of substitution on the dispersibility of the complex.
The amphiphilic peptides were chemically synthesized by Fmoc solid-phase synthesis method. The products were purified using HPLC and identified by MALDI TOF-MS. The complex between peptides and Ptx was prepared by mixing an aqueous solution containing the peptides as well as Cu(II) and an ethanol solution of Ptx, followed by removal of ethanol in vacuo and lyophilization. To the complex was added 0.10 mL phosphate buffer solution to evaluate of the apparent solubility of Ptx. When Ptx was complexed with the peptide alone, the dispersibility of Ptx was not significantly different from that of the blank sample. The apparent solubility increased with increasing the concentration of Cu(II). The dispersibility of Ptx complex using KHHA3WA3 and KHHA6W was comparable to that using KHHA7, suggesting that the effect of substitution to tryptophan on the complex stability is small.

[SC]PL229 Characteristics of human adipose-derived stem cells on 2D and 3D cultures
Department of Life and Environment Engineering, The University of Kitakyushu, Fukuoka, JAPAN

Mesenchymal stem cells including adipose-derived stem cells (ADSCs) are expected as a cell source for tissue engineering and regenerative medicine, because they can be differentiated to osteoblasts, chondrocytes, adipocytes, and so on. 2D monolayer culture that cells are extended on a culture substrate has been widely used as a conventional culture technique. On the other hand, 3D culture, such as spheroid (spherical organoid) and cell-sheet, has attracted attention as a new culture technique. In this study, we investigated the difference of ADSC properties in the monolayer (2D) and spheroid (3D) cultures.
As a spheroid formation platform for 3D culture, we fabricated a microwell which consisted of 195 circular microwells (400μm in diameter) on a poly-methylmethacrylateplate, and the surface was modified by polyethylene glycol to promote the spheroid formation. Traditional tissue culture plate was used as a monolayer culture platform for 2D culture. The cell proliferation and differentiation properties of ADSCs were compared between monolayer and spheroid cultures.
In the 2D culture, the cells adhered and extended on the culture plate, and proliferated during culture period. In the 3D culture, the cells were aggregated in each microwell and formed the spheroid within 6 hours of culture. The spheroid morphology was maintained in each microwell. However, the size of ADSC spheroids was almost same during culture period, and the cell proliferation of spheroid culture was suppressed than that of monolayer culture. The expression levels of differentiation marker genes (osteoblast, chondrocyte, and adipocyte differentiations) in the spheroid culture were higher than those in monolayer culture.
These results indicate that the proliferation and differentiation of ADSCs are influenced by the difference of cell morphology, and the spheroid culture is a promising method to promote the cell differentiation.

[SC]PL230 Drug responses of cancer cell spheroids in a microwell chip culture
Hanaka Kozono, Daisuke GO, Kohji NAKAZAWA
The University of Kitakyushu

In development processes of anti-cancer drugs, cell-based assays have been used as one of drug screening techniques. Generally, monolayer culture of cancer cells has been utilized as in cancer model, but it is difficult to mimic morphology and environments of in vivo neoplasm. On the other hand, cancer cell spheroids, which are formed by the rearrangement and compaction of cell aggregates, have attracted attention as more useful solid cancer model. We have developed a spheroid culture platform, microwell chip, which can achieve size control, easy medium change, and spheroid mass production. In this study, we investigated the difference of drug responses of cancer cells in the monolayer and spheroid cultures.
The microwell chip consisted of 200 circular microwells (600 μm in diameter) on a poly-dimethylsiloxane plate, and the surface was modified by polyethylene glycol to promote the spheroid formation. Traditional tissue culture plate was used as a monolayer culture. HCT116 cells (colorectal cancer cell line) and doxorubicin (anti-cancer drug) were used as the cancer model cell and model drug, respectively. The cells were treated with different doxorubicin concentrations, and the changes in cell morphology and cell survival were evaluated in the both culture platforms.
HCT116 cells formed a single spheroid in each microwell within 3 days of culture. The spheroid growth was inhibited by the doxorubicin treatment, and the collapse phenomenon at outside region of spheroids occurred by the doxorubicin treatment of high concentration. In the monolayer culture, the cells were detached from culture plate with the increase of doxorubicin concentration. The cell survival rate against doxorubicin in spheroid culture was higher than that in monolayer culture, indicating that HCT116 spheroids have high drug resistance than monolayer culture.
These results suggested that spheroid culture has a potential as cell-based assay technique for drug screening.

[SC]PL231 Theranostics with hybrid liposomes in orthotopic graft model mice of breast cancer
Sojo University, Kumamoto, Japan

Hybrid liposomes (HL) composed of L-α-dimyristylphosphatidylcholine (DMPC) and polyoxyethylene (25) dodecyl ether (C12(EO)25) were effective for therapeutics and detection for tumor of orthotopic graft model mouse of breast cancer.
In the therapeutic effects of HL for breast cancer, high inhibitory effects of HL without drugs on the growth of breast cancer (MDA-MB-453) cells were obtained along with apoptosis in vitro. Membrane fluidity of MDA-MB-453 cells treated with HL was significantly increased as compared with that of control and DMPC liposomes. We examined therapeutic effects of HL for orthotopic graft model mice of breast cancer after the inoculation to mammary gland. Reduction of tumor in the group treated with HL was observed, although enlargement of tumor in control and DMPC groups was confirmed on the basis of volume, weight and autopsy. Many apoptotic cells were observed in the tumor section of orthotopic graft model mice of breast cancer treated with HL using TUNEL method.
With regard to detection (diagnosis) on breast cancer using HL, enhanced selective accumulation (diagnosis) of HL/ICG including a fluorescence probe (Indocyanine green; ICG) only toward breast cancer cells having high membrane fluidity without affecting normal breast cells was confirmed in vitro. Enhanced accumulation of HL/ICG into the tumor of orthotopic graft model mice of breast cancer was observed into the tumor of orthotopic graft model mice of breast cancer was observed. These data indicate that HL accumulated in tumor cells in the mammary gland of the orthotopic graft mouse model of breast cancer for a prolonged period of time, and inhibited the growth of MDA-MB-453 cells.
Therapeutic effects by HL and detection (diagnosis) by HL/ICG of cancer with enhanced tumor accumulation for orthotopic graft model mouse of MDA-MB-453 for the use of HL as theranostics agents were revealed for the first time in vivo.

[SC]PL232 Therapeutic effects and antitumor mechanism of trehalose liposomes against breast adenocarcinoma.
SOJO University, Kumamoto, Japan

Specific binding on liposome of the trehalose with a high hydration power has been reported. Trehalose liposomes (DMTre) have been produced by using sonication of a mixture of L-α-dimyristoylphosphatidylcholine (DMPC) and α-D-glycopyranosyl-α-D-glucopiranoside monomyristate (TreC14) in buffer solutions with no organic solvent. Inhibitory effects of trehalose liposomes on the growth of tumor cells along with apoptosis have been reported. Growth inhibition against colon, gastric, hepatocellular and leukemia cells has been obtained using trehalose liposomes without drugs.In this study, we investigated inhibitory effects of trehalose liposomes on the proliferation of human breast adenocarcinoma ( MDA-MB-453 ) cells were examined in vitro and in vivo. DMTre with a hydrodynamic diameter less than 100 nm, were preserved for 4 weeks. The inhibitory effects of DMTre on the proliferation of MDA-MB-453 cells accompanied with apoptosis were obtained after the enhancement of accumulation of DMTre into MDA-MB-453 cell membranes. An increase in membrane fluidity of MDA-MB-453 cells treated with DMTre was observed on the basis of fluorescence depolarization method. DMTre suppressed the activity of NFホコB. DMTre caused apoptosis for MDA-MB-453 cells through the activation of caspases and mitochondria. Suppression on the filopodia formation of MDA-MB-453 cell by DMTre was observed using confocal laser microscopy. Inhibitory effects of DMTre on the invasion of MDA-MB-453 cells was also obtained using invasion assay. Tumor weights on the xenograft model mice of MDA-MB-453 intravenously administered with DMTre markedly decreased as compared with those of the control group. Brown color indicating apoptotic cells was observed in many tumor cells of tissue section of tumor in xenograft model mice after the treatment with DMTre. DMtre suppressed metastasi in hepatic metastasis mouse model of breast tumor after the intrasplenic inoculation of MDA-MB-453 cells. Therapeutic effect of DMtre were also obtained using hepatic metastasis mouse model of breast tumor.

[SC]PL233 Synthesis and characterization of micro/nano liquid metal complexes for the drug delivery applications
Dasom KIM1, Jangsun HWANG1, Yonghyun CHOI1, Kyobum KIM2, Hyung-Jun KOO3, Jonghoon CHOI1
1 School of Integrative Engineering, Chung-Ang University, Seoul 06974, Republic of Korea
2 Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea
3 Department of Chemical and Biomolecular Engineering, Seoul National University of Science and Technology, Seoul 01811, Republic of Korea

Among various drug delivery systems, micro/nanocarriers consist of liquid metal are being actively studied. Eutectic Gallium-Indium alloy (EGaIn) liquid metal has been reported to have a low melting point, low toxicity, and proper tissue/cell penetration. EGaIn has a characteristic property, deforming by the control of light and heat irradiations. In this study, we describe transformable liquid metal nanoparticles delivering anti-tumor drug molecules. These “nanotransformer” particles are synthesized through sonication, and extrusion of either EGaIn solution or EGaIn mixed with amphiphilic lipids. The resulting nanotransformers are loaded with an anti-tumor drug, doxorubicin that can be released upon deformation of EGaIn nanoparticles excited with light. The nanotransformer particles showed no toxicity in mammalian cells. A proper photo-thermal ligand added to the nanotransformer helped to increase the yield of deformed liquid metal nanoparticles, resulting in efficient drug delivery.

[SC]PL234 Facial fabrication of size-controllable vesicles using micromixer in microfluidic chip
Bongsu KANG1, Shogo TAGUCHI2, Keesung KIM4, Younghawn SON5, Moonkyu KWAK1, Hosup JUNG3
1 School of Mechanical Engineering, Kyungpook Nat. Univ, Daegu, Republic of Korea
2 Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, Osaka, Japan
3 Center for Food and Bioconvergence, Dept. of Food Science and Biotechnology, Seoul Nat. Univ, Seoul, Republic of Korea
4 Research Institute of Advanced Materials, Collage of Engineering, Seoul Nat. Univ, Seoul, Republic of Korea
5 Department of landscape architecture and rural system engineering, Seoul National University, Seoul, Republic of Korea

A phospholipid forms a self-assembled membrane such as micelles, bicelles and vesicles to reduce an exposure of hydrophobic chain groups in aqueous solution. Interestingly, bicelles are disk-shaped aggregates composed of long-chain phospholipids that make up a planar region and either detergent or short-chain phospholipids that compose flanking rims. They have an advantage over micelles in their ability to mimic natural membranes and, therefore, capture membrane proteins in their biologically relevant orientation as demonstrated in structural studies of the transmembrane segment, functional studies of membrane protein and drug delivery system.
Previously, we reported that as 窶彙icelles窶・ are made of comprise a mixture of long-chained phospholipids (e.g., 1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC) and short-chained phospholipids (e.g., 1,2-dihexanoyl-sn-glycero-3-phosphocholine, DHPC) at high concentration in aqueous solution, wherein DHPC detergents cover the rim of DMPC bilayer. In addition, bicelles can be reformed vesicles depend on a various condition such as total lipid concentration and DMPC/DHPC fraction ratios. However, it is very difficult to control of vesicle size by using simple flow microfluidic chip.
In this study, we demonstrate simple method to control the size of vesicles through dilution of bicelles solution by using microfluidic chip which has integrated with passive micromixer and microchannel structure to produce the flow focusing. The results shows that the final production is formed vesicle structure of ordered phase through fusion of bicelles. In addition, we verified the membrane properties with the membrane fluidity (1/P_DPH), polarity (GP340) and optical density (OD500). Our novel microfluidic chip has potentials to create a lipid vesicles which can be controlled size by using total flow rate ratio and DMPC/DHPC fraction ratio.

[SC]PL235 Screening of CdTe quantum dot-binding peptides for quantum dot-antibody bioconjugation
Thanawat SUWATTHANARAK1, Daisuke MINAMIDE1, Masayoshi TANAKA1, Andrew HARVIE2, Abiral TAMANG2, Kevin CRITCHLEY2, Stephen EVANS2, Mina OKOCHI1
1 Tokyo Institute of Technology, Tokyo, Japan
2 University of Leeds, Leeds, United Kingdom

Quantum dots (QDs) are promising fluorescent probes for many biological applications because they have superior and unique optical properties in comparison to conventional organic dyes. Traditionally, covalent coupling is employed to immobilize or conjugate antibodies onto QD surface for preparation of QD-antibody immunoprobes. In this study, we proposed a simple and effective technique to conjugate CdTe QD with immunoglobulin G (IgG) antibody using high-affinity bifunctional peptides. First, five CdTe QD-binding peptides were obtained from phage display screening. To identify the shortest amino acid sequence needed for the QD-binding activity, the screened peptides were subjected to truncation assay using SPOT-peptide array. Linking the truncated peptides with an antibody Fc domain-binding peptide1 was later done to design mediate bifunctional peptides for bioconjugation of CdTe QD with IgG antibody. The affinities of bifunctional peptides to CdTe QD and IgG antibody were confirmed by surface plasmon resonance (SPR) system. Due to no extensive surface preparation steps and chemicals as covalent conjugations, the designed bifunctional peptides suggest simply and biological-friendly conjugation process of CdTe QD with IgG antibody. Our peptide-mediated molecule conjugation strategy would be beneficial in development of QD and antibody-based molecular imaging.
1Sugita, T. et al. Screening of peptide ligands that bind to the Fc region of IgG using peptide array and its application to affinity purification of antibody. Biochem Eng J. 2013, 79, 33–40.

[SC]PL236 Microengineering of collagen hydrogels integrated into microfluidic devices for perfusion culture of mammalian cells
Misaki KATO, Mayu FUKUSHI, Masumi YAMADA, Rie UTOH, Minoru SEKI
Chiba University, Chiba City, Chiba, Japan

Engineering of collagen-based hydrogels in microfluidic devices is a promising technology for cell culture experiments with well-controlled environmental parameters. However, it is not a trivial task to integrate microfabricated, cell-encapsulating hydrogels into microfluidic devices. Here we present a new approach to precisely engineering type-I collagen-based hydrogels in microfluidic devices, with the help of gelation agent-incorporating composite silicone substrates. We created microposts composed of polydimethylsiloxane (PDMS) and phosphate salt (Na2HPO4) powders, which work as a neutralizer for acidic collagen solution into collagen hydrogels selectively around the microposts. It was possible to encapsulate living mammalian cells into the hydrogels, which could be extended to perfusion culture of cells for organs-on-a-chip applications.
In the experiments, we fabricated PDMS microfluidic devices equipped with microposts with various morphologies, by soft lithography and replica molding techniques. After introducing an acidic collagen solution into the channel and incubating the solution for several minutes, washing buffer was introduced to remove the non-gelled collagen solution from the channel. As a result, we successfully fabricated collagen hydrogels selectively around the microposts (see the attached figure). Additionally, we were able to create vascular tissue models, when we employed a microchannel whose lumen was completely made of the composite PDMS substrate containing phosphate salt powders. Living mammalian cells could be encapsulated in the hydrogel matrices while keeping high cell viability, showing its applicability to perfusion cell culture. The presented strategy would be highly useful because we could fabricate collagen hydrogel-based microstructures in microfluidic devices without the need for complicated devices or operations.

[SC]PL237 Fabrication of bilirubin chip for neonatal hyperbilirubinemia diagnosis
Yen-Ching TAI, Jen-Huang HUANG
National Tsing Hua University, Hsinchu, Taiwan

Neonatal hyperbilirubinemia, which is also called neonatal jaundice is a common symptom in the newborn. Bilirubin accumulates in the newborns' blood and high concentration of bilirubin is potentially toxic to the central nervous system, so it's critical to monitor the level of bilirubin. Current bilirubin diagnosis is still based on diazotization reaction and calibrated with the standards using an automated clinical chemistry analyzer to obtain the bilirubin value. The total measuring time is about 30 min. However, most of the diagnosis has to be completed in large hospitals or medical centers due to the costly diagnosis equipment. For those who lives in remote areas or are lack of medical resources, it's impossible to obtain immediate medical diagnosis.
In this work, we combine the advantages of paper-based diagnosis technology, the efficiency of the active separation process, and the aspects of the microfluidic platform to construct a bilirubin diagnosis chip which can measure the values of total, direct and indirect bilirubin. The diagnosis time of bilirubin chip can be controlled within 5 min and the requirement of sample volume is only one drop of the blood. The samples can be quantitatively analyzed by using the bilirubin chip directly and the colorimetric readout which can be obtained through a smartphone camera without using the expensive and bulky equipment. Moreover, we will establish a standard operating procedure for neonatal bilirubin testing and apply it directly to clinical trial. After the comparison of the clinical chemistry analyzer and transcutaneous bilirubin meter, we can establish a real time, easily operate, and precise bilirubin testing system to meet the goal of point-of-care diagnosis.

[SC]PL238 Correlation between maintaining stemness of MSCs grown on NWF scaffold and ECM
Bo FU, Masashi FUJIWARA, Mutsumi TAKAGI
Hokkaido University, Sapporo, Japan

Mesenchymal stem cells (MSCs) have been ideal candidates for cell therapy because of extensive proliferative ability and stemness. We tried to use nonwoven fabrics (NWF) as a cell culture scaffold. NWF has three-dimensional fiber aggregates formed by heat bonding, which has a high surface area for cell adhesion and elongation. Although NWF have been reported to be a candidate scaffold for large scale expansion of MSCs, quality of cells grown in NWF was not known well. In this report, MSCs grown in a disc scaffold of NWF for 3 weeks showed higher osteogenic differentiation potential and percentage of CD90 positive cells, compared with MSCs grown in a dish bottom surface. Amount of extracellular matrix (ECM) per unit surface area of fibers was larger than that on a bottom surface of dish at first 2 weeks of culture. The osteogenic differentiation potential of the MSC inoculated onto cell-free ECM-coated dish was higher as the amount of ECM increased. Consequently, the higher percentage of CD90 positive cell and higher osteogenic differentiation potential of cells grown in a NWF disc than cells cultured on a dish might at least partly due to the higher amount of ECM.
Keywords: mesenchymal stem cell, nonwoven fabric, ECM, CD90, osteogenic differentiation

[SC]PL239 Preparation of nanostructured multi-phase lipid career for triggered release under intestinal condition
Ren OHGAKI1, Takashi Kobayashi1, Kyuya NAKAGAWA2
1 Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Japan
2 Department of Chemical Engineering, Faculty of Engineering, Kyoto University, Kyotodaigaku-Katsura, Nishikyo-Ku, Kyoto 615-8510 Japan

Nanostructured multi-phase lipid career (NMLC) is recognized as potential delivery vehicle for sensitive biological compounds. It has been reported that the drug release behavior from NMLC is affected by the type of lipids, emulsifiers and conditions employed for preparation. In this study, multi-phase lipid particles were prepared by hot shear homogenization method with wax and triglyceride mixture. This particulate system is designed to release compounds dissolved in the triglyceride phase by the reaction of intestinal enzyme (i.e. lipase). The carnauba wax, distilled water and coconut oil were homogenized (15,000 rpm, 100°C 5 seconds) and cooled rapidly at 0°C Samples with different ratio of coconut oil and wax, addition of emulsifier (i.e. sorbitan monooleate, sodium cholate), and different cooling temperature (i.e. 0°C and 30°C were prepared. The particle size and surface structure were observed with a scanning electron microscope (SEM). The NMLC dispersion was reacted with simulated intestinal fluid (i.e. lipase, maleic acid buffer (pH 6.5), 37°C . After the reaction, the amounts of the free fatty acids (FFA) produced by the coconut oil were measured by neutralization titration. The particle size of the prepared NMLCs was in the range of 2–100 ホシm. Formation of the channel of coconut oil could be observed on the surface of the particle. When these NMLCs were applied to the intestinal fluid, FFA were successfully released from the NMLCs. The released amounts of FFA were similar for the samples without emulsifiers. This was because the coconut oil phase exposed on the particle surface did not differ largely among the samples. The amounts of FFA formation were greatly increased in the samples with the emulsifiers. It found that the emulsifier would help the lipase reaction, which accelerates the hydrolysis of coconut oil.

[SC]PL240 Adsorption and Desorption Behavior of Drugs on a QCM Sensor-supported Apatite
Kitami Institute of Technology

Availability of quartz crystal microbalance (QCM) technique as screening method of drug–loaded ceramic material was examined. Hydroxyapatite {apatite; Ca10(PO4)6(OH)2} was coated on a QCM sensor with wet synthesizing or self-assembled monolayer (SAM)-mediating procedure. Adsorption and desorption behavior of two anticancer reagents: mitoxantrone dihydrochloride (MXT) and doxorubicin hydrochloride (DOX) were followed on the QCM sensor-supported apatite. The 80 ホシL reagent of 1g/L-distilled water was injected into 8 mL water-containing glass cup where the sensor tip had been immersed. The decreased frequency (ホ認dec) in injection was about 300 for MXT or 500 Hz for DOX, which indicated adsorption of both reagents occurred in agreement with the order of the adsorbed amounts on apatite particle.
After he frequency (F) became constant for about 1 h, phosphate buffered solution (PBS, pH 7) of 100 mmol/L was injected. MXT and DOX showed opposite profiles. For MXT, the F increased in injection, indicating that desorption occurred possibly due to substituting MXT by ions in PBS on the surface. On the contrary, the F for DOX did not increase but did decrease more after the constant F, i.e., adsorption in pseudo-equilibrium. This result was much different from that for apatite particle, in which desorption of DOX was clearly observed in a batch system. Dependency of the injected concentration of PBS (0 – 100 mmol/L) on the ホ認dec was examined. As the concentration increased, the ホ認dec became larger, suggesting that PBS facilitated adsorption of DOX as opposed to MXT.
The Zeta potentials of MXT and DOX were much different: MXT was almost zero while DOX was highly-positively charged: about 23 mV. From these results, we considered that negatively–charged phosphate ion in PBS bound to positively-charged DOX and then less-charged DOX adsorbed on the surface, with possibly weak binding such as van der Waals adsorption.