Dal-Hee Min

Prospects and Challenges of Graphene in Biomedical Applications

Graphene materials have entered a phase of maturity in their development that is characterized by their explorative utilization in various types of applications and fields from electronics to biomedicine. Herein, we describe the recent advances made with graphene-related materials in the biomedical field and the challenges facing these exciting new tools both in terms of biological activity and toxicological profiling in vitro and in vivo. Graphene materials today have mainly been explored as components of biosensors and for construction of matrices in tissue engineering. Their antimicrobial activity and their capacity to act as drug delivery platforms have also been reported, however, not as coherently. This report will attempt to offer some perspective as to which areas of biomedical applications can expect graphene-related materials to constitute a tool offering improved functionality and previously unavailable options.

Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes: proliferation, focal adhesion, and gene transfection studies.

Carbon-based materials, including graphene and carbon nanotubes, have been considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for stem cell differentiation, and components of implant devices. Despite the potential biomedical applications of these materials, only limited information is available regarding the cellular events, including cell viability, adhesion, and spreading, that occur when mammalian cells interface with carbon-based nanomaterials. Here, we report behaviors of mammalian cells, specifically NIH-3T3 fibroblast cells, grown on supported thin films of graphene and carbon nanotubes to investigate biocompatibility of the artificial surface. Proliferation assay, cell shape analysis, focal adhesion study, and quantitative measurements of cell adhesion-related gene expression levels by RT-PCR reveal that the fibroblast cells grow well, with different numbers and sizes of focal adhesions, on graphene- and carbon nanotube-coated substrates. Interestingly, the gene transfection efficiency of cells grown on the substrates was improved up to 250% that of cells grown on a cover glass. The present study suggests that these nanomaterials hold high potential for bioapplications showing high biocompatibility, especially as surface coating materials for implants, without inducing notable deleterious effects while enhancing some cellular functions (i.e., gene transfection and expression).

Facile Synthesis of Monodispersed Mesoporous Silica Nanoparticles with Ultralarge Pores and Their Application in Gene Delivery

Among various nanoparticles, the silica nanoparticle (SiNP) is an attractive candidate as a gene delivery carrier due to advantages such as availability in porous forms for encapsulation of drugs and genes, large surface area to load biomacromolecules, biocompatibility, storage stability, and easy preparation in large quantity with low cost. Here, we report on a facile synthesis of monodispersed mesoporous silica nanoparticles (MMSN) possessing very large pores (>15 nm) and application of the nanoparticles to plasmid DNA delivery to human cells. The aminated MMSN with large pores provided a higher loading capacity for plasmids than those with small pores (∼2 nm), and the complex of MMSN with plasmid DNA readily entered into cells without supplementary polymers such as cationic dendrimers. Furthermore, MMSN with large pores could efficiently protect plasmids from nuclease-mediated degradation and showed much higher transfection efficiency of the plasmids encoding luciferase and green fluorescent protein (pLuc, pGFP) compared to MMSN with small pores (∼2 nm).

A Graphene‐Based Platform for the Assay of Duplex‐DNA Unwinding by Helicase

Time to unwind: Graphene oxide (GO) enables the quantitative measurement of helicase‐dependent double‐stranded DNA (dsDNA) unwinding activity in real time. GO selectively binds to unwound fluorescent‐dye‐labeled single‐stranded DNA and quenches its fluorescence (see picture). The helicase activity is monitored by following the change in fluorescence.WILEY-VCH

Quantitative and Multiplexed MicroRNA Sensing in Living Cells Based on Peptide Nucleic Acid and Nano Graphene Oxide (PANGO)

MicroRNA (miRNA) is an important small RNA which regulates diverse gene expression at the post-transcriptional level. miRNAs are considered as important biomarkers since abnormal expression of specific miRNAs is associated with many diseases including cancer and diabetes. Therefore, it is important to develop biosensors to quantitatively detect miRNA expression levels. Here, we develop a nanosized graphene oxide (NGO) based miRNA sensor, which allows quantitative monitoring of target miRNA expression levels in living cells. The strategy is based on tight binding of NGO with peptide nucleic acid (PNA) probes, resulting in fluorescence quenching of the dye that is conjugated to the PNA, and subsequent recovery of the fluorescence upon addition of target miRNA. PNA as a probe for miRNA sensing offers many advantages including high sequence specificity, high loading capacity on the NGO surface compared to DNA and resistance against nuclease-mediated degradation. The present miRNA sensor allowed the detection of specific target miRNAs with the detection limit as low as ~1 pM and the simultaneous monitoring of three different miRNAs in a living cell.

Durable Large-Area Thin Films of Graphene/Carbon Nanotube Double Layers as a Transparent Electrode

We prepared durable, uniform, large-area ultrathin transparent films composed of double layers of reduced graphene oxide (RG-O) and multiwalled carbon nanotubes (MWNTs) via a self-assembly process. The adsorption of MWNTs onto RG-O films considerably decreased the sheet resistance of the films without compromising much on transparency. This self-assembly approach could be used to fabricate transparent electronic devices without post-transfer processes on a large scale.

Synergistic Effect of Graphene Oxide/MWCNT Films in Laser Desorption/Ionization Mass Spectrometry of Small Molecules and Tissue Imaging

Matrix-assisted laser desorption/ionization mass spectrometry has been considered an important tool for various biochemical analyses and proteomics research. Although addition of conventional matrix efficiently supports laser desorption/ionization of analytes with minimal fragmentation, it often results in high background interference and misinterpretation of the spatial distribution of biomolecules especially in low-mass regions. Here, we show design, systematic characterization, and application of graphene oxide/multiwalled carbon nanotube-based films fabricated on solid substrates as a new matrix-free laser desorption/ionization platform. We demonstrate that the graphene oxide/multiwalled carbon nanotube double layer provides many advantages as a laser desorption/ionization substrate, such as efficient desorption/ionization of analytes with minimum fragmentation, high salt tolerance, no sweet-spots for mass signal, excellent durability against mechanical and photoagitation and prolonged exposure to ambient conditions, and applicability to tissue imaging mass spectrometry. This platform will be widely used as an important tool for mass spectrometry-based biochemical analyses because of its outstanding performance, long-term stability, and cost effectiveness.

Efficient functional delivery of siRNA using mesoporous silica nanoparticles with ultralarge pores.

Among various nanoparticles, mesoporous silica nanoparticles (MSNs) have attracted extensive attention for developing efficient drug-delivery systems, mostly due to their high porosity and biocompatibility. However, due to the small pore size, generally below 5 nm in diameter, potential drugs that are loaded into the pore have been limited to small molecules. Herein, a small interfering RNA (siRNA) delivery strategy based on MSNs possessing pores with an average diameter of 23 nm is presented. The siRNA is regarded as a powerful gene therapeutic agent for treatment of a wide range of diseases by enabling post-transcriptional gene silencing, so-called RNA interference. Highly efficient, sequence-specific, and technically very simple target gene knockdown is demonstrated using MSNs with ultralarge pores of size 23 nm in vitro and in vivo without notable cytotoxicity.

A New Assay for Endonuclease/Methyltransferase Activities Based on Graphene Oxide

A new endonuclease/methyltransferase activity assay method based on graphene oxide (GO) is developed. Substrate DNA is designed to possess a double-stranded part to serve as a nuclease substrate and a single-stranded part for anchoring the DNA to the GO surface via strong noncovalent binding. Nuclease-mediated DNA hydrolysis induces the recovery of fluorescence intensity of the dye attached to the end of the double-stranded DNA region. This GO-based method allows real-time measurement and quantitative assay for endonuclease/methyltransferase activities in short time.

Influence of Surface Functionalization on the Growth of Gold Nanostructures on Graphene Thin Films

We developed a surface-chemistry-based approach to investigating the influence of surface functionalization on the growth of gold nanostructures on graphene thin films by utilizing various pyrene derivatives presenting different functional groups. Among the surface-modifying molecules, decylpyrene (DP) yielded the highest content of gold rods (average 22 +/- 4%) among gold nanostructures on graphene films when a graphene surface was pretreated with DP prior to gold nanostructure growth. The improved yield of gold rods on graphene thin films enhanced several physical properties of graphene such as the electrical conductivity and Raman signals by 6.3- and 14.7-fold, respectively.