Hyeon-Yeol Cho

Simultaneous capture and in situ analysis of circulating tumor cells using multiple hybrid nanoparticles

Using hybrid nanoparticles (HNPs), we demonstrate simultaneous capture, in situ protein expression analysis, and cellular phenotype identification of circulating tumor cells (CTCs). Each HNP consists of three parts: (i) antibodies that bind specifically to a known biomarker for CTCs, (ii) a quantum dot that emits fluorescence signals, and (iii) biotinylated DNA that allows capture and release of CTC-HNP complex to an in-house developed capture & recovery chip (CRC). To evaluate our approach, cells representative of different breast cancer subtypes (MCF-7: luminal; SK-BR-3: HER2; and MDA-MB-231: basal-like) were captured onto CRC and expressions of EpCAM, HER2, and EGFR were detected concurrently. The average capture efficiency of CTCs was 87.5% with identification accuracy of 92.4%. Subsequently, by cleaving the DNA portion with restriction enzymes, captured cells were released at efficiencies of 86.1%. Further studies showed that these recovered cells are viable and can proliferate in vitro. Using HNPs, it is possible to count, analyze in situ protein expression, and culture CTCs, all from the same set of cells, enabling a wide range of molecular- and cellular-based studies using CTCs.

In situ monitoring of doxorubicin release from biohybrid nanoparticles modified with antibody and cell-penetrating peptides in breast cancer cells using surface-enhanced Raman spectroscopy.

In situ monitoring of drug release in cancer cells is very important for real-time assessment of drug release dynamics in chemotherapy. In this study, we report label-free in situ monitoring and control of intracellular anti-cancer drug delivery process using biohybrid nanoparticles based on surface-enhanced Raman spectroscopy (SERS) for the first time. Each biohybrid nanoparticle consisted of gold nanoparticle, cell-penetrating peptide (Tat peptide), and cancer-targeting antibody to increase the efficacy of the anti-cancer drug delivery with specific targeting and increased uptake rate. The doxorubicin (Dox)-loaded biohybrid nanoparticles were showed specific SERS spectra of Dox, specifically immobilized on the target cell membrane and quickly penetrated into the cells when treated on the mixed cell culture condition. The intracellular release of Dox from the biohybrid nanoparticle was continuously monitored with time-dependent change of intracellular SERS signals of Dox. The releasing rate of Dox was successfully controlled with the addition of glutathione on the cells. The anti-cancer effect of intracellular released Dox was confirmed with cell viability assay. With the proposed monitoring system, specific cancer cell targeting and improved uptake of the anti-cancer drug were detected and time-dependent intracellular release of the anti-cancer drug was monitored successfully. The proposed novel in situ monitoring system can be used as a spectroscopic analysis tool for label-free monitoring of the time-dependent release of various kinds of anti-cancer drugs inside cells.

Engineered peptide-based nanobiomaterials for electrochemical cell chip

Biomaterials having cell adhesion ability are considered to be integral part of a cell chip. A number of researches have been carried out to search for a suitable material for effective immobilization of cell on substrate. Engineered ECM materials or their components like collagen, Poly-l-Lysine (PLL), Arg-Gly-Asp (RGD) peptide have been extensively used for mammalian cell adhesion and proliferation with the aim of tissue regeneration or cell based sensing application. This review focuses on the various approaches for two- and three-dimensionally patterned nanostructures of a short peptide i.e. RGD peptide on chip surfaces together with their effects on cell behaviors and electrochemical measurements. Most of the study concluded with positive remarks on the well-oriented engineered RGD peptide over their homogenous thin film. The engineered RGD peptide not only influences cell adhesion, spreading and proliferation but also their periodic nano-arrays directly influence electrochemical measurements of the chips. The electrochemical signals found to be enhanced when RGD peptides were used in well-defined two-dimensional nano-arrays. The topographic alteration of three-dimensional structure of engineered RGD peptide was reported to be suitably contacted with the integrin receptors of cellular membrane which results indicated the enhanced cell-electrode adhesion and efficient electron exchange phenomenon. This enhanced electrochemical signal increases the sensitivity of the chip against the target analytes. Therefore, development of engineered cellular recognizable peptides and its 3D topological design for fabrication of cell chip will provide the synergetic effect on bio-affinity, sensitivity and accuracy for the in situ real-time monitoring of analytes.

Cell chip with a thiolated chitosan self-assembled monolayer to detect the effects of anticancer drugs on breast normal and cancer cells

Cell-based chips are an effective in vitro analysis tool; however, the sensitivity of the cell chip to biomaterials is high, which is crucial for immobilizing cells on the electrode surface without conductivity. In this study, we report on a cell chip with a thiolated chitosan monolayer that was easy to fabricate, highly adhesive to cells, and enhanced electrochemical signals. Thiolated chitosan containing thiol groups was synthesized and self-assembled on a gold electrode to immobilize cells, and showed superior electrochemical performance to that of poly-l-lysine and collagen. Cyclic voltammetry (CV) was performed to distinguish the redox characteristics of normal (HMEC) and breast cancer cells (MCF-7); then, two anticancer drugs (doxorubicin and cyclophosphamide) were added to the cell cultures to analyze their effects on the redox environment of normal and cancer cells derived from the same origin. As a result, the CV cathode peaks decreased differently with respect to the cell line (normal and cancer) and anticancer drug, which was validated by a conventional MTT viability assay. Hence, the proposed cell chip with a thiolated chitosan modified layer could be used in various fields, including discriminating normal from cancer cells, to evaluating the efficiency of newly developed drugs, and to assessing cytotoxicity of various chemicals.

Electrically Controlled Delivery of Cargo into Single Human Neural Stem Cell

Nanoprobe-based techniques have emerged as an efficient tool for the manipulation and analysis of single cells. Here, we report a powerful whole-electrical single-cell manipulation tool that enables rapid and controllable delivery of cargo into single neural stem cells with precision monitoring of the cell penetration process using a conductive nanoprobe. The highly electrically sensitive nanoprobes that were fabricated and the indium tin oxide electrode-integrated cell chip were found to be very effective for monitoring the cell penetration process via current changes that appear as spike-like negative currents. Moreover, the assembly of cargoes onto the nanoprobes was controllable and could reach its maximum load in a very short period of time (<10 min) based on the same electrical system that was used for monitoring cell penetration and without the need for any complex chemical linkers or mediators. Even more remarkably, the cargo assembled on the surface of the nanoprobe was successfully released in a very short period of time (<10 s), regardless of the surrounding intracellular or extracellular environments. The monitoring of cell penetration, assembly of quantum dots (QDs), and release of QDs into the intracellular environment were all accomplished using our whole-electrical system that combined a conductive nanoprobe with cell chip technology. This is a novel technology, which can eliminate complex and time-consuming steps owing to chemical modifications, as well as reduce the time needed for the delivery of cargo into the cell cytosol/nucleus during cell penetration, which is very important for reducing cell damage.

Magnetic Oleosome as a Functional Lipophilic Drug Carrier for Cancer Therapy

In the present study, we fabricated magnetic oleosomes functionalized with recombinant proteins as a new carrier for oil-based lipophilic drugs for cancer treatment. The bioengineered oleosome is composed of neutral lipids surrounded by a phospholipid monolayer with embedded oleosin fusion proteins. The oleosin was genetically fused to a nanobody of a green fluorescent protein (GFP). A recombinant protein consisting of immunoglobulin-binding protein LG fused to GFP was used to couple the oleosome to an antibody for targeted delivery to breast cancer cells. The lipid core of the oleosome was loaded with magnetic nanoparticles and carmustine as the lipophilic drug. The magnetic oleosome was characterized using transmission electron microscopy and dynamic light scattering. Moreover, the specific delivery of oleosome into the target cancer cell was investigated via confocal microscopy. To examine the cell viability of the delivered oleosome, a conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was carried out. Furthermore, an animal study was conducted to confirm the effect resulting from the delivery of the anticancer drug-loaded oleosomes. Taken together, the fabricated lipophilic drug-loaded magnetic oleosome can be a powerful tool for oil-based drug delivery agent for cancer therapy.

Neural Cell Chip Based Electrochemical Detection of Nanotoxicity

Development of a rapid, sensitive and cost-effective method for toxicity assessment of commonly used nanoparticles is urgently needed for the sustainable development of nanotechnology. A neural cell with high sensitivity and conductivity has become a potential candidate for a cell chip to investigate toxicity of environmental influences. A neural cell immobilized on a conductive surface has become a potential tool for the assessment of nanotoxicity based on electrochemical methods. The effective electrochemical monitoring largely depends on the adequate attachment of a neural cell on the chip surfaces. Recently, establishment of integrin receptor specific ligand molecules arginine-glycine-aspartic acid (RGD) or its several modifications RGD-Multi Armed Peptide terminated with cysteine (RGD-MAP-C), C(RGD)4 ensure farm attachment of neural cell on the electrode surfaces either in their two dimensional (dot) or three dimensional (rod or pillar) like nano-scale arrangement. A three dimensional RGD modified electrode surface has been proven to be more suitable for cell adhesion, proliferation, differentiation as well as electrochemical measurement. This review discusses fabrication as well as electrochemical measurements of neural cell chip with particular emphasis on their use for nanotoxicity assessments sequentially since inception to date. Successful monitoring of quantum dot (QD), graphene oxide (GO) and cosmetic compound toxicity using the newly developed neural cell chip were discussed here as a case study. This review recommended that a neural cell chip established on a nanostructured ligand modified conductive surface can be a potential tool for the toxicity assessments of newly developed nanomaterials prior to their use on biology or biomedical technologies.

Application of Gold Nanoparticle to Plasmonic Biosensors

Gold nanoparticles (GNPs) have been widely utilized to develop various biosensors for molecular diagnosis, as they can be easily functionalized and exhibit unique optical properties explained by plasmonic effects. These unique optical properties of GNPs allow the expression of an intense color under light that can be tuned by altering their size, shape, composition, and coupling with other plasmonic nanoparticles. Additionally, they can also enhance other optical signals, such as fluorescence and Raman scattering, making them suitable for biosensor development. In this review, we provide a detailed discussion of the currently developed biosensors based on the aforementioned unique optical features of GNPs. Mainly, we focus on four different plasmonic biosensing methods, including localized surface plasmon resonance (LSPR), surface-enhanced Raman spectroscopy (SERS), fluorescence enhancement, and quenching caused by plasmon and colorimetry changes based on the coupling of GNPs. We believe that the topics discussed here are useful and able to provide a guideline in the development of novel GNP-based biosensors in the future.

Subtyping of Magnetically Isolated Breast Cancer Cells Using Magnetic Force Microscopy

Circulating tumor cell (CTC) which recently arisen as potential sources for monitoring and characterizing non-haematologic cancers and their metastatic derivatives. Immunomagnetic microbeads and magnetic nanoparticles (MNPs) have been extensively explored to isolate CTCs from blood samples. However, MNPs attached on the membrane protein are interrupted further analysis to distinguish the cancer subtype by consumption or blocking the target surface marker. Here, an MNP-mediated analysis method for surface marker expression profile by magnetic force microscopy (MFM) is described. Two MNPs, zinc ferrite and iron oxide, are showed distinct phase shift (-16.5° and -3.7°, respectively) signal on the MFM images. The antibody conjugated MNPs are successfully isolated target cells without giving damage to the cell. The MFM image of MNP decorated cells show clear differences between two breast cancer cell lines, MCF-7 and SK-BR-3, which proof the cancer subtyping property using MFM method. To confirmation of the surface marker consumption during the cell isolation, antibody-conjugated quantum dots and drug-loaded oleosome are treated on the cells, thereby MNP decorated cells are survived. This newly developed MFM analysis method provides a new direction to utilize the MNP for the surface marker expression phenotypes.

Overcoming Chemoresistance in Cancer via Combined MicroRNA Therapeutics with Anticancer Drugs Using Multifunctional Magnetic Core–Shell Nanoparticles

In this study, we report the use of a multifunctional magnetic core-shell nanoparticle (MCNP), composed of a highly magnetic zinc-doped iron oxide (ZnFe2O4) core nanoparticle and a biocompatible mesoporous silica (mSi) shell, for the simultaneous delivery of let-7a microRNA (miRNA) and anticancer drugs (e.g., doxorubicin) to overcome chemoresistance in breast cancer. Owing to the ability of let-7a to repress DNA repair mechanisms (e.g., BRCA1 and BRCA2) and downregulate drug efflux pumps (e.g., ABCG2), delivery of let-7a could sensitize chemoresistant breast cancer cells (MDA-MB-231) to subsequent doxorubicin chemotherapy both in vitro and in vivo. Moreover, the multifunctionality of our MCNPs allows for the monitoring of in vivo delivery via magnetic resonance imaging. In short, we have developed a multifunctional MCNP-based therapeutic approach to provide an attractive method with which to enhance our ability not only to deliver combined miRNA therapeutics with small-molecule drugs in both selective and effective manner but also to sensitize cancer cells for the enhanced treatment via the combination of miRNA replacement therapy using a single nanoplatform.