Development of Bio-Hybrid Nanoparticles based In Situ Raman Imaging for Intracellular miRNA Detection

Currently, microRNAs (miRNAs) were widely recognized as valuable biomarkers to identify in typical cancer characteristics. These small RNA molecules frequently exhibited dysregulation in various cancers, leading them to serve as either oncogenic or tumor-suppressor miRNAs, depending on their specific target genes. Moreover, miRNAs can modulate cancer cells to chemotherapy. Real time polymerase chain reaction (RT-PCR) was currently used as a method to detect intracellular miRNA. However, it required cell lysis, miRNA isolation, purification that makes hard to use in live cells. Therefore, several fluorescence-based analysis techniques (e.g., fluorescence resonance energy transfer (FRET)) were used for live cell detection, but they had limitation in detecting miRNA with low expression levels. We aimed to overcome these limitations by using surface-enhanced Raman spectroscopy (SERS), a technique widely used as a tool for sensing application, which analyzed based on the distances between molecules, to detect intracellular miRNA in live cells.<br/>In this study, we developed highly sensitive Raman activating gold nanoparticles (HS-RGNs) which were containing gold nanoparticles, Raman reporter, and single strand DNAs (ssDNAs) complementary to target miRNA. When HS-RGNs hybridized with target miRNA, these particles amplified the Raman signal. We selected microRNA 200c (miR-200c) as a model biomaterial which was related to the chemoresistance of cancer cells. After treatment of HS-RGNs to SK-BR-3 and MCF-7 which are cancer cells, the ssDNAs of HS-RGN were hybridized with miR-200c, bringing two HS-RGNs closer, thereby enhancing the Raman signal. The higher expression level of miR-200c in SK-BR-3 compared to MCF-7 led to the observation of a higher Raman signal in SK-BR-3 cells. When treated with HS-RGNs, increased expression levels of Zinc finger E-box binding homeobox 1 (Zeb1) and Tropomyosin receptor kinase B (TrkB), which were inhibited by miR-200c, indicated the effective binding of HS-RGNs to miR-200c. Additionally, when assessing the cytotoxicity of doxorubicin, SK-BR-3, which exhibited higher miR-200c expression, showed a lower survival rate compared to MCF-7. However, when treated with HS-RGNs, miR-200c was effectively consumed, resulting in a higher survival rate in both SK-BR-3 and MCF-7 cells. This result suggested that HS-RGNs can hybridize with miR-200c, impact of miRNA on cell behavior.<br/>Our study showed that HS-RGNs effectively amplified the Raman signal, indicating their potential applicability for in situ intracellular analysis. Additionally, they can be applied as a platform technology to investigate the impact of miRNA on cell behavior by consuming miRNA within cells.<br/><br/>Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00211360) and Biomaterials Specialized Graduate Program through the Korea Environmental Industry & Technology Institute(KEITI) funded by the Ministry of Environment(MOE).<br/><br/>Reference<br/>[1] W.-J. Lee, K.-J. Kim, M.K. Hossain, H.-Y. Cho, J.-W. Choi, DNA–Gold Nanoparticle Conjugates for Intracellular miRNA Detection Using Surface-Enhanced Raman Spectroscopy, BioChip Journal 16(1) (2022) 33-40.