Yu-Chung Lin

Nanodiamond for intracellular imaging in the microorganisms in vivo.

Nanodiamond (ND) has great potential for bio labeling and drug delivery. In this work, the biocompatibility and bio labeling of ND are demonstrated via the interaction with cells and microorganisms, protists microorganisms Paramecium caudatum and Tetrahymena thermophile, in vitro and in vivo. We found the microorganisms living functions are not significantly affected by ND. The NDs were found entering the food vacuoles and later excreted by the microorganisms. The 5 nm ND was found more toxic compared to 100 nm ND, presumably due to the surface disordered carbons. Our results demonstrated nanodiamond can be used in bio imaging and matter delivery.

The influence of nanodiamond on the oxygenation states and micro rheological properties of human red blood cells in vitro

Abstract. Nanodiamond has been proven to be biocompatible and proposed for various biomedical applications. Recently, nanometer-sized diamonds have been demonstrated as an effective Raman/fluorescence probe for bio-labeling, as well as, for drug delivery. Bio-labeling/drug delivery can be extended to the human blood system, provided one understands the interaction between nanodiamonds and the blood system. Here, the interaction of nanodiamonds (5 and 100 nm) with human red blood cells (RBC) in vitro is discussed. Measurements have been facilitated using Raman spectroscopy, laser scanning fluorescence spectroscopy, and laser diffractometry (ektacytometry). Data on cell viability and hemolytic analysis are also presented. Results indicate that the nanodiamonds in the studied condition do not cause hemolysis, and the cell viability is not affected. Importantly, the oxygenation/deoxygenation process was not found to be altered when nanodiamonds interacted with the RBC. However, the nanodiamond can affect some RBC properties such as deformability and aggregation in a concentration dependent manner. These results suggest that the nanodiamond can be used as an effective bio-labeling and drug delivery tool in ambient conditions, without complicating the blood’s physiological conditions. However, controlling the blood properties including deformability of RBCs and rheological properties of blood is necessary during treatment.

Nanodiamond for biolabelling and toxicity evaluation in the zebrafish embryo in vivo

Nanodiamond (ND) has been proposed for various biomedical applications, including bioimaging, biosensing and drug delivery, owing to its physical-chemical properties and biocompatibility. Particularly, ND has been demonstrated as fluorescence- and Raman-detectable labels in many cellular models. Different surface functionalization methods have been developed, varying the NDs surface properties and rendering the possibility to attach biomolecules to provide interaction with biological targets. For this, toxicity is of major concern in animal models. Aside from cellular models, a cost-effective animal test will greatly facilitate the development of applications. In this study, we use the rapid, sensitive and reproducible zebrafish embryo model for in vivo nanotoxicity test. We optimize the conditions for using this animal model and analyze the zebrafish embryonic development in the presence of ND. ND is observed in the embryo in vivo using laser confocal fluorescence microscopy and fluorescence lifetime imaging. Using the zebrafish model for a safety evaluation of ND-based nanolabel is discussed.

Nanodiamonds for Medical Applications: Interaction with Blood in Vitro and in Vivo

Nanodiamonds (ND) have emerged to be a widely-discussed nanomaterial for their applications in biological studies and for medical diagnostics and treatment. The potentials have been successfully demonstrated in cellular and tissue models in vitro. For medical applications, further in vivo studies on various applications become important. One of the most challenging possibilities of ND biomedical application is controllable drug delivery and tracing. That usually assumes ND interaction with the blood system. In this work, we study ND interaction with rat blood and analyze how the ND surface modification and coating can optimize the ND interaction with the blood. It was found that adsorption of a low concentration of ND does not affect the oxygenation state of red blood cells (RBC). The obtained in vivo results are compared to the results of in vitro studies of nanodiamond interaction with rat and human blood and blood components, such as red blood cells and blood plasma. An in vivo animal model shows ND injected in blood attach to the RBC membrane and circulate with blood for more than 30 min; and ND do not stimulate an immune response by measurement of proinflammatory cytokine TNF-α with ND injected into mice via the caudal vein. The results further confirm nanodiamonds’ safety in organisms, as well as the possibility of their application without complicating the blood’s physiological conditions.

Raman spectroscopic study on the excystation process in a single unicellular organism amoeba (Acanthamoeba polyphaga)

Abstract. An in vivo Raman spectroscopic study of amoeba (Acanthamoeba polyphaga) is presented. The changes of the spectra during the amoeba cyst activation and excystation are analyzed. The spectra show the changes of the relative intensities of bands corresponding to protein, lipid, and carotenoid components during cyst activation. The presence of carotenoids in the amoeba is observed via characteristic Raman bands. These signals in the Raman spectra are intense in cysts but decrease in intensity with cyst activation and exhibit a correlation with the life cycle of amoeba. This work demonstrates the feasibility of using Raman spectroscopy for the detection of single amoeba microorganisms in vivo and for the analysis of the amoeba life activity. The information obtained may have implications for the estimation of epidemiological situations and for the diagnostics and prognosis of the development of amoebic inflammations.

Spectral Analysis of Nanodiamond-Berberine Complex Interaction with Living Cells for Nanoparticle Mediated Drug Delivery

The natural isoquinoline alkaloid berberine has been demonstrated for its significant activity against a variety of deceases caused by bacterial and parasite infections. Nanodiamond (ND) has also been proven to have great potential for drug delivery. In this work, the complex of ND with berberine is constructed through surface functionalized ND, aiming for drug delivery applications. Spectroscopic analysis was performed on the complex interaction with human lung alveolar carcinoma epithelial cell (A549) and red blood cell (RBC) in comparison with berberine in solution. The studied cells were treated with carboxylated ND, ND-berberine complex and berberine, respectively. Their distributions in cells structures are visualized using ND and berberine’s fluorescence signals for detection. The distribution of ND and ND-berberine complex differs with the distribution of berberine introduced in solution form. The results show that the ND with attached drug molecules can act as a drug carrier allowing controllable localization of the drug into the cell. This result can have implication in drug delivery, release and delivery tracing applications.