課題番号 ：S-16-NM-0055
利用形態 ：機器利用
利用課題名（日本語） ：
Program Title (English) ：Development of a novel immune adjuvant nanoparticle
利用者名（日本語） ：
Username (English) ：Hwan-Hee OH
所属名（日本語） ：
Affiliation (English) ：JSPS

１．概要（Summary ）
Development of a novel immune adjuvant nanoparticle: embossing surface structure using tethered ODNs for immune disease therapy
-Preparation of phospholipid-ODN conjugated nano particle
-Evaluation of stimulated immune responses
The development of a novel immune adjuvant is emerging as a significant issue of drug delivery to amplify immune response for allergic diseases, infectious disease and cancer therapy. It has been reported that oligodeonynucleotides (ODNs) containing unmethylated cytosine-phosphate-guanine (CpG) motifs can activate the immune response with interaction of pattern-recognition receptor Toll-like receptor 9 (TLR9). To stimulate immune response and increasing efficiency of uptake, various NPs combining with ODNs containing CpG and free-CpG motifs were preliminarily studied in our laboratory. In this regard, I am designing a novel NPs with embossing surface structured of ODNs for improvement of ODN uptake efficiency, high immune response stimulation and innovation of ODN delivery.

Figure 1. Schematic diagram of the embossing ODN structured NPs. Representative image of a) ss-ODN, As-ssODN and phospholipid, b) conjugated ss-ODN and As-ss ODN with phospholipid, c) hybridization to dsODN d) and e) packing and formation of micelle structured NPs.

２．実験（Experimental）
RT-PCR: to measure cytokine release after cell culture with fabricated nanoparticle including ODN.
Gradient-PCR: to prepare double strand ODN for further experiment.
Cell culture facility: to investigation of cytokine release
FT-IR: To confirm the spectrum of synthetic material
DLS analyzer: to investigate the size of nanoparticle
Zeta potential measurement: to confirm the surface charge of modified nanoparticle
SEM: to reveal the structure of nanoparticle
Freeze-dryer: to fabricate nanoparticle

３．結果と考察（Results and Discussion）

1) Characterization of ODN-PE and hybridization
ODNs with 3’-sulfhydryls were conjugated to maleimide-PE (sulfhydryl-reactive maleimide group conjugated to the lipid head group of PE) through the formation of a thioether bond. In order to ensure that the enhanced immuno-pharmacological effects were not due to contaminating free ODNs, the PE-ODN conjugates were purified using size-exclusion chromatography (SEC) to remove unconjugated ODN. Fractions from size-exclusion chromatography were confirmed using TG-gel.
Based on the result of electrophoresis, PE conjugated ODN showed at the high molecular area and unconjugated ODN was located on 24bp area. It might be caused from hydrophobic interaction of aliphatic chain in PE. Identified fractions of PE-ODN was collected and used for further research. The synthesized PE-ODNs (PE-AS CpG-ODN and PE-AS nonCPG ODN) were hybridizied with single-stranded ODNs (CpG-ODN, nonCpG-ODN) for double strand ODN.
Single stranded ODNs were successfully hybridized and evaluated by using TG gel with SYBR green II staining. As we expected, the double stranded ODNs including PE lipid were appeared at high molecular area in TG gel. Free single stranded ODNs (Figure 2. b and g) were not appeared after hybridization. From result of electrophoresis, I confirmed that the PE conjugated ODNs were successfully synthesized, purified and hybridized. The two different types of nanoparticles were used to enhance cell uptake and escape from endosome. DOTAP is a liposome of the cationic lipid. DOTAP can be used for the highly efficient transfection of negatively charged molecules. And the other is amorphous calcium phosphate (ACP) prepared by using a soluble phosphate and calcium salt. The hydrodynamic diameter size of compound NPs with ODNs was confirmed by DLS measurement.

2) Evaluation of compound NPs with ODNs The complex of NPs and ODNs were evaluated for their ability to induce the secretion of the Th1-type proinflammatory cytokine IFN-beta. Four different types of dsODNs were electrostatically bound on two different NPs including ACP and DOTAP. After 6 hours incubation with complex of NP and ODN, lysate of culture cell were collected and used for RT-PCR
A significant higher induction of INF-beta was confirmed in complex of NPs bound with PE conjugated dsODN. This enhanced induction phenomenon was not related to CpG motif in ODNs but, with PE conjugation. In conclusion, I introduced preparation of PE lipid conjugated with ODN for development of a novel adjuvant. I successfully synthesized the PE lipid with ODNs by using emulsion conjugation method and conjugated PE-ODNs were also successfully purified by chloroform washing and size-exclusion chromatography. The result of chloroform washing was confirmed with TLC silica gel. Factions of size-exclusion chromatography and hybridization of ODNs were examined with TG gel electrophoresis. After electrostatic binding on NPs, PE conjugated ds CpG-ODN and ds non CpG-ODN showed enhanced induction of IFN-beta than ds CpG and ds non CpG. Extension of this PE modified ODN system enables to create the opportunity to develop new applications in he fields of biomedical and biomaterials

４．その他・特記事項（Others）
Facility staffs assisted me to use instruments. Also assisted me on data interpretation.

５．論文・学会発表（Publication/Presentation）
なし。

６．関連特許（Patent）
なし。