Yun Mi Bae

Long-Term Real-Time Tracking of Lanthanide Ion Doped Upconverting Nanoparticles in Living Cells†

Recently, there has been great interest in employing nanoparticles for various biological applications. Nanoparticles can be synthesized in a controlled manner such that they have desirable sizes, shapes, and optical or magnetic properties. In addition, one may provide nanoparticles with biological functions through chemical surface modifications and conjugation of ligands. Such intrinsic and extrinsic properties of nanoparticles enable them to be used as excellent biological imaging probes and diagnostic/therapeutic agents at the cellular level. Among the various nanoparticle systems developed thus far, semiconductor nanocrystals or quantum dots (QDs) are most widely used. QDs are extremely bright and photostable, and exhibit excellent spectral properties (i.e., broad absorption and narrow emission bands) suited for multicolor detection. However, the drawbacks such as photoblinking, the presence of nonradiant dark particles, and potential cytotoxicity limit their applicability. In recent years, several alternative types of luminescent nanoparticles have been introduced for biological applications. For example, nanodiamonds (NDs) with nitrogen vacancy centers were found to be highly photoluminescent while exhibiting no photoblinking and photobleaching, 11] and even useful as the imaging probe for super-resolution optical microscopy. However, applying NDs for biological imaging has limitations, especially in the case of long-term tracking studies, since the excitation in the blue or green region (typically 488 or 532 nm) might result in fatal photodamage to cells or low penetration depth into tissues. In contrast, single-walled carbon nanotubes (SWNTs) were shown to be appropriate for biological imaging in that the excitation and emission lie in the near-infrared (NIR) spectral range. However, being longer than 100 nm typically, SWNTs are considered to be too large to be used as biolabels. Meanwhile, lanthanide ion doped upconverting nanoparticles (UCNPs), which emit in the visible range upon absorption of NIR photons, have attracted great attention owing to their unique optical properties. First, two-photon upconversion of NIR excitation to the emission of a visible photon is so efficient that a tiny continuous-wave (CW) diode laser (980 nm) with the output of tens of milliwatts is sufficient as the excitation source. Second, by employing NIR excitation, one can suppress cellular autofluorescence, induce little photodamage to living cells, and achieve relatively deep penetration into tissues. Finally, UCNPs exhibit neither photoblinking on the millisecond and second time scales nor photobleaching even with hours of continuous excitation, 21] their cytotoxicity is very low, 22] and the inclusion or doping of Gd ions in the host materials endows UCNPs with an additional modality for magnetic resonance imaging (MRI). 23] As a result, UCNPs became one of the most promising nanoparticle systems for biological imaging and there are continuing efforts to improve their properties (e.g., increasing luminescence intensity and reducing the particle size) by designing new synthetic strategies. Herein, we report the first real-time tracking study with UCNPs at the single vesicle level in living cells. Thanks to the remarkable photostability of UCNPs and the noninvasiveness of NIR excitation, we were able to visualize the intracellular movements of UCNPs for as long as 6 h without interruption. We first assessed the benefits of using NIR radiation as the excitation source to demonstrate the feasibility of long-term live-cell imaging with UCNPs. The UCNPs (hexagonal-phase NaYF4 co-doped with Yb 3+ and Er, ca. 30 nm in diameter) coated by amphiphilic PEG–phospholipids (PEG = poly(ethylene glycol)) were internalized into HeLa cells and imaged on a home-made epi-fluorescence microscope setup (Methods section and Figures S1 and S2 in the Supporting [*] Dr. S. H. Nam, Y. M. Bae, Dr. H. M. Kim, Dr. K. T. Lee, Dr. Y. D. Suh Laboratory for Advanced Molecular Probing (LAMP) NanoBio Fusion Research Center, Korea Research Institute of Chemical Technology, Daejeon 305-600 (Korea) Fax: (+ 82)42-860-7164 E-mail: ktlee@krict.re.kr ydsuh@krict.re.kr Y. I. Park, Dr. J. H. Kim, Prof. Dr. T. Hyeon National Creative Research Initiative Center for Oxide Nanocrystalline Materials, World Class University (WCU) Program of Chemical Convergence for Energy & Environment (C2E2) School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744 (Korea) Y. M. Bae, Prof. Dr. J. S. Choi Department of Biochemistry, Chungnam National University Daejeon 305-764 (Korea) [] These authors contributed equally to this work.

Synthesis of PAMAM Dendrimer Derivatives with Enhanced Buffering Capacity and Remarkable Gene Transfection Efficiency

In this study, we introduced histidine residues into l-arginine grafted PAMAM G4 dendrimers to enhance proton buffering capacity and evaluated the physicochemical characteristics and transfection efficacies in vitro. The results showed that the synthesized PAMAM G4 derivatives effectively delivered pDNA inside cells and the transfection level improved considerably as the number of histidine residues increased. Grafting histidine residues into the established polymer vector PAMAM G4-arginine improved their proton buffering capacity. The cytotoxicity of PAMAM G4 derivatives was tested and it was confirmed that they displayed relatively lower cytotoxicity compared to PEI25KD in various cell lines. Also, confocal microscopy results revealed that PAMAM G4 derivatives effectively delivered pDNA into cells, particularly into the nucleus. These PAMAM dendrimer derivatives conjugated with histidines and arginines may provide a promising polymeric gene carrier system.

Dexamethasone-conjugated polyethylenimine as an efficient gene carrier with an anti-apoptotic effect to cardiomyocytes.

Dexamethasone is a potent glucocorticoid with anti‐inflammatory effects. Dexamethasone can protect ischemic cardiomyocytes from apoptosis. To apply the anti‐apoptotic effect of dexamethasone to ischemic disease gene therapy, dexamethasone‐conjugated polyethylenimine (PEI‐Dexa) was synthesized and evaluated as an anti‐apoptotic gene carrier.

Preparation of orthogonally functionalized surface using micromolding in capillaries technique for the control of cellular adhesion

This study presents a simple method for the fabrication of an orthogonal surface that can be applied for cell patterning without the need to immobilize specific adhesive peptides, proteins, or extracellular matrix (ECM) for cell attachment. Micromolding in capillaries (MIMIC) produced two distinctive regions. One region contained poly(ethylene glycol)-poly(D,L-lactide) diblock copolymer (PEG-PLA) designed to provide a biological barrier to the nonspecific binding of proteins and fibroblast cells. The other region was coated with polyelectrolyte (PEL) to promote the adhesion of biomolecules including proteins and cells. Resistance to the adsorption of proteins increased with the length of PEG and PLA chains because the longer PEG chain increased the PEG layer thickness and the longer PLA chain induced stronger interaction with the PEL surface. The PEG5k-PLA2.5k (20mg/ml) was the most efficient candidate for the prevention of protein adhesion among the PEG-PLA copolymers examined. The orthogonal functionality of prepared surfaces having PEL regions and background PEG-PLA regions resulted in rapid patterning of biomolecules. Fluorescein isothiocyanate-tagged bovine serum albumin (FITC-BSA) and fibroblast cells successfully adhered to the exposed PEL surfaces. Although methods for cell patterning generally require an adhesive protein layer on the desired area, these fabricated surfaces without adhesive proteins provide a gentle microenvironment for cells. In addition, our proposed approach could easily control patterns, sizes, and shapes at micron scale.

Enhanced splicing correction effect by an oligo-aspartic acid–PNA conjugate and cationic carrier complexes

Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA. They recognize specific cellular nucleic acid sequences and form stable complexes with complementary DNA or RNA. Here, we designed an oligo-aspartic acid-PNA conjugate and showed its enhanced delivery into cells with high gene correction efficiency using conventional cationic carriers, such as polyethylenimine (PEI) and Lipofectamine 2000. The negatively charged oligo-aspartic acid-PNA (Asp(n)-PNA) formed complexes with PEI and Lipofectamine, and the resulting Asp(n)-PNA/PEI and Asp(n)-PNA/Lipofectamine complexes were introduced into cells. We observed significantly enhanced cellular uptake of Asp(n)-PNA by cationic carriers and detected an active splicing correction effect even at nanomolar concentrations. We found that the splicing correction efficiency of the complex depended on the kind of the cationic carriers and on the number of repeating aspartic acid units. By enhancing the cellular uptake efficiency of PNAs, these results may provide a novel platform technology of PNAs as bioactive substances for their biological and therapeutic applications.

Amino acid-modified bioreducible poly(amidoamine) dendrimers: Synthesis, characterization and In vitro evaluation

AbstractPoly(amidoamine) (PAMAM) dendrimers are synthetic polymers commonly used as carriers in gene and drug delivery. PAMAMs have the ability to transfect DNA, but their transfection efficiency is currently insufficient for clinical use. Here, we demonstrate the synthesis and evaluation of cationic dendrimers consisting of a cystamine core PAMAM generation 3 (cPAM G3) and amino acids. Introduction of histidine (His) and arginine (Arg) residues to cPAM G3 resulted in high transfection efficiency and low cytotoxicity. cPAM G3-His-Arg formed stable polyplexes at a weight ratio of 6:1, and the mean polyplex diameter was 99.03±1.68 nm. Nano-sized polyplexes increased in diameter up to 132.93±1.79 nm when treated with a reducing agent, dithiothreitol (DTT). cPAM G3-His-Arg showed much higher transfection efficiency than native cPAM G3 and polyethylenimine (PEI, 25KD). In addition, cPAM G3-His-Arg displayed negligible toxicity, even at high polymer concentrations. Finally, confocal laser microscopy results showed that cPAM G3-His-Arg effectively internalized plasmid DNA into the cells. Therefore, we believe that cPAM G3-His-Arg could be a promising bioreducible vector for non-viral gene delivery with high gene transfection efficiency and low cytotoxicity.

Preparation of cationic liposome containing a novel water-soluble detergent and its application to gene deliveryIn vitro

As gene delivery carrier vectors, various synthetic nonviral vectors have been developed over the past decade. The synthetic vehicles for encapsulation and delivery of therapeutic genes are cationic liposomes and polymers, or the combination of both vectors. Some recent studies show that they express sufficient amounts of therapeutically active compounds in distal tumors, causing reduced tumor growth. For the synthetic carriers studied so far, liposome system is one of the most promising carriers for delivering therapeutic genes as well as biologically active molecules into mammalian cells. However, low efficiency of synthetic nonviral gene carriers including liposome has been the major limitation for their successful clinical applications. In addition, for liposomal gene delivery systems, the stability problem of the liposome as well as liposome/DNA complex has been raised for systemic gene delivery and the “Stealth” liposomal system concept was introduced for the efficient liposomal drug/gene delivery applications. As well as the problems in physicochemical properties of the liposomal gene carriers, the membranous structure of cells also gave the main barriers for gene delivery using nonviral carriers. Here, we report the novel cationic detergent-containing liposomal system and its application to gene delivery in vitro. The cationic detergent contains hydroxyl functional groups on its hydrophilic group, which provide the potential conjugation sites for introducing multifuntionality such as, TAT-derived peptides, membrane-disrupting peptides, or nucleus targeting signals.