2022 Fiscal Year Research-status Report
Creation and biological evaluation of nanoparticle-based artificial transcription factor
| Project/Area Number |
22K15249
|
|---|
| Research Institution | Kyoto University |
|---|
Principal Investigator |
|
|---|
| Project Period (FY) |
2022-04-01 – 2024-03-31
|
| Keywords | Transcription factor / Silica nanoparticles / Mitochondrial factors / Cell Imaging / Molecular modeling / medaka egg imaging |
|---|
| Outline of Annual Research Achievements |
Silicon quantum dots (SiQDs) capable of in vitro and in vivo imaging could dissect cellular function at the molecular level and have been advancing as a potent diagnostic tool in precision medicine. While numerous reports focus on evaluating the staining capacity of silica quantum dots, their nano-bio interaction, which is critical for assessing their biocompatibility, is often overlooked. We developed water-borne, NIR-emitting Pluronic F127-covered SiQDs micelles capable of live cell imaging with minimal cytotoxicity. The PLQY of the SiQDs/F127 micelles in water was 25% at pH 7. SiQD-De/F127 micelles demonstrated significant in vitro imaging capacity and limited cytotoxicity in the HeLa and human dermal fibroblasts. Furthermore, the SiQD-De/F127 micelles displayed excellent in vivo imaging capacity without requiring vectors and transfection agents when incubated with medaka fish eggs and larvae. It is important to note here that even at higher concentrations (1 mg mL－1), the normal embryonic development of medaka fish was not altered as the larvae hatched on day 17 from the micelles-treated egg as that of the control without any signs of toxicity. Therefore, SiQDs-De/F127 micelles are SiQDs expected to have excellent biocompatibility for in vivo imaging studies.
|
| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
An artificial transcription factor will be developed based on silica nanoparticles as a carrier, enabling more accurate ROS transcriptional control in cancer cells. In this process, we prepared fluorescent mesoporous silica nanoparticles with sizes varying from 50 to 200 nm. Additionally, we analyzed nanomaterials characterization using equipment like transmission electron microscopy, dynamic light scattering, etc. Recently, carbon quantum dots have been used for mitochondrial penetrating experiments because of their size. Therefore, we have collaborated with a Malaysian university to synthesize carbon quantum dots from natural sources like palm shells and oysters.
|
| Strategy for Future Research Activity |
We plan to use various nanomaterials to prepare the NANO-TF, including silica and carbon quantum dots. Simultaneously process Nu- and Mito-NANO-TF to investigate whether AMPK pathway-related genes in the nucleus and ROS production-related genes in mitochondria can be controlled in stages. First, analyze the intracellular ROS level by staining with 2'-7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) and flow cytometry to investigate the ROS level control ability of NANO-TF. Quantify the expression of marker proteins such as PGC-1α using Western blot, and investigate cancer cell death by cell viability assay and flow cytometer analysis.
|
| Causes of Carryover |
In the next fiscal year, we plan to do the biological evaluation of stepwise regulation of the AMPK pathway by NANO-TF. Next-generation sequencing analysis will be performed using biotin-labeled NANO-TF and AMPK pathway-related mitochondrial signaling pathways and essential genes to search and identify signal motifs that control the expression of biotin. Simultaneously process Nu- and Mito-NANO-TF to investigate whether AMPK pathway-related genes in the nucleus and ROS production-related genes in mitochondria can be regulated in stages. First, analyze the intracellular ROS level by staining with 2'-7'-Dichlorodihydrofluorescein diacetate and flow cytometry to investigate the ROS level control ability of NANO-TF. Quantify the expression of marker proteins such as PGC-1α using Western blot, and investigate cancer cell death by cell viability assay and flow cytometer analysis. In addition, I need to purchase chemicals for organic synthesis, antibody, and enzyme assay reagents, sequencing analysis reagents, reagents for cell culture, Staining kits, Tips, and glassware. To participate in domestic and international conferences.
|
