Jimmy K. Li

Synthesis, Characterization, and Bioconjugation of Fluorescent Gold Nanoclusters toward Biological Labeling Applications

Synthesis of ultrasmall water-soluble fluorescent gold nanoclusters is reported. The clusters have a decent quantum yield, high colloidal stability, and can be readily conjugated with biological molecules. Specific staining of cells and nonspecific uptake by living cells is demonstrated.

Design of an amphiphilic polymer for nanoparticle coating and functionalization.

Inorganic colloidal nanoparticles, such as quantum dots or Au nanoparticles, have been extensively investigated for two decades in physics as well as in chemistry. Applications in a variety of fields such as optics, electronics, and biology are envisaged and important proof-of-concept studies have been reported. In particular, with regard to biologically motivated applications, colloidal stability is a key requirement. Apart from nanoparticles stabilized with small ligand molecules, lipids, [6–8] and surface silanization, amphiphilic polymers have been also used by several groups to disperse originally hydrophobic nanoparticles in aqueous solution. This class of amphiphilic particle coatings not only enables the phase transfer of the nanoparticles from organic solvents to aqueous solution, but also serves as a versatile platform for chemical modification and bioconjugation of the particles because biological molecules can be covalently linked to the polymer surface. Because the stability of the amphiphilic coating around the nanoparticle solely depends on the hydrophobic interaction, this procedure is very general and does, for example, not depend on the material of the inorganic nanoparticle core, as it is the case for ligand exchange protocols. Because of the numerous contact points mediated by hydrophobic interaction, the attachment of the polymer to the particle surface is highly stable and can be improved further by crosslinking of the polymer shell. Nowadays quantum dots coated with amphiphilic polymers and with various biological molecules attached to their surface are commercially available (e.g., Invitrogen). The amphiphilic polymers that have been used so far for coating hydrophobic inorganic nanoparticles consist of hydrophobic side chains for the linkage to the nanoparticle surface and a hydrophilic backbone that provides water solubility through charged groups (in general -COO ) and also acts as an anchor for the attachment of biological molecules with bioconjugate chemistry. In this report, we introduce an amphiphilic polymer which involves a third kind of building block: functional organic molecules. The functional organic molecules are linked to the hydrophobic side chains in a similar way as the hydrophilic backbone and provide additional functionality in the particle surface (Figure 1). The amphiphilic polymer described here is based on a poly(maleic anhydride) backbone. Reaction of a fraction of the anhydride rings with alkylamines leads to the formation of the hydrophobic side chains that are needed for intercalation with the hydrophobic surfactant layer on the nanoparticle surface. Another fraction of the anhydride rings is used to link functional organic molecules to the backbone. Like the alkylamines, organic molecules bearing amino-groups can be directly linked to the anhydride rings by reaction of the anhydride with the amino group. In this way alkylamines and organic molecules with amino terminations can be linked to the polymer backbone in a one-pot reaction. The resulting amphiphilic polymer is then wrapped around hydrophobic capped nanoparticles and the organic solvent is replaced by aqueous solution according to our previously published procedure. By linking some of the remaining anhydride rings with diamine linkers, the polymer molecules around each nanoparticle are interconnected and, thus, the shell is crosslinked. Upon phase transfer to aqueous solution, the remaining anhydride rings open to yield negatively charged carboxyl groups, which provide electrostatic repulsion resulting in a stable dispersion of the nanoparticles. Apart from negatively charged carboxyl groups, the polymer surface of the nanoparticles also contains embedded functional organic molecules. The strategy reported here has several advantageous features: 1) The maleic anhydride moieties react spontaneously with high yield with both amino-modified hydrophobic side-chains (such as alkylamines) and functional organic molecules with amino terminal groups. 2) No additional reagents are needed for the coupling. In comparison, [*] R. A. Sperling, M. Zanella, Prof. W. J. Parak Fachbereich Physik, Philipps Universit#t Marburg Renthof 7, 35037 Marburg (Germany) E-mail: Wolfgang.Parak@physik.uni-marburg.de C.-A. J. Lin, R. A. Sperling, P.-Y. Li, M. Zanella, Prof. W. J. Parak Center for NanoScience Ludwig-Maximilians-Universit#t M8nchen Munich (Germany) C.-A. J. Lin, T.-Y. Yang, W. H. Chang Department of Biomedical Engineering Chung Yuan Christian University Taiwan (ROC) C.-A. J. Lin, J. K. Li, W. H. Chang R&D Center for Membrane Technology Center for Nano Bioengineering Chung Yuan Christian University Taiwan (ROC) [] These authors contributed equally to this work. [] Present address: Institute of Biotechnology, National Cheng Kung University, Taiwan (ROC)

Cytokine release from osteoblasts in response to ultrasound stimulation

Bone is a dynamic tissue with a well-balanced homeostasis preserved by both formation and resorption of bone. Normal turnover of bone, however, can be upset by either increased osteoclast activity or decreased osteoblast function; either mechanism alone or both may result in a net loss of bone. Both osteoclasts and osteoblasts could be stimulated by mechanical stimulation in vitro, and it is assumed that this process may occur in vivo as well. In this experiment, we investigated this hypothesis by examining the effects of ultrasound stimulation on osteoblast growth and cytokine release. With this model, we explored the mechanism of low-intensity pulsed ultrasound on osteoblasts growth and upregulation of osteoclasts formation and function by cytokine release. The results showed that specific pulsed ultrasound exposure could enhance osteoblasts population together with increase in TGFbeta1 secretion and decrease in concentration of IL-6 and TNFalpha in the culture medium. Although, animal studies and clinical trial are needed to understand the real process in the whole body, ultrasound stimulation might be a good method for prevention of bone loss due to osteoporosis.

Cytokine Release from Osteoblasts in Response to Different Intensities of Pulsed Electromagnetic Field Stimulation

We use an in-vitro osteoblast cell culture model to investigate the effects of low-frequency (7.5 Hz) pulsed electromagnetic field (PEMF) stimulation on osteoblast population, cytokines (prostaglandin E2 (PGE2), transforming growth factor β1(TGFβ1), and alkaline phosphatase (ALP) activity to find the optimal intensity of PEMF for osteoblast growth. The results demonstrate that PEMF can stimulate osteoblast growth, release of TGFβ1, and, in addition, an increase of ALP activity. The synthesis and release of PGE2 in the culture medium are reduced with increasing numbers of cells. Higher intensity does not necessarily mean increased osteoblast growth, and the most efficient intensity is about 2 mV/cm in this case. Although the lower intensities of the PEMF are yet to be determined, the results of this study can shed light on the mechanisms of PEMF stimulation on non union fracture therapy and osteoporosis prevention in the future.

Effects of Ultrasound on Osteotomy Healing in a Rabbit Fracture Model

This study investigated the effects of ultrasound (US) at different frequencies on fracture healing over a three-week period in a rabbit fibular fracture model. Forty-five adult New Zealand White rabbits were divided into five groups: a control group and four groups treated with US frequencies of 0.5, 1.0, 1.5 and 2.0 MHz (0.5 W/cm(2), 200-μs burst, pulsed 1:4). After anesthesia, transverse osteotomy was performed on the fibula bone. This was followed by intravital staining and fluorescence microscopic examination of new bone formation and biomechanical tests of torsional stiffness at the osteotomy site. Results showed that total new bone formation and torsional stiffness of the fibula were greater in all US-treated groups than in the control group. No significant difference was found between any of the four US-treated groups. The US treatment also enhanced bone growth of the sham-treated contralateral fracture site. These results suggest that US treatment at 0.5, 1.0, 1.5 or 2.0 MHz can enhance fracture healing in a rabbit model. Furthermore, the effects of US on fracture healing at present parameters might not be confined locally.

Synthesis and surface modification of highly fluorescent gold nanoclusters and their exploitation for cellular labeling

We introduce a general approach to make colorful fluorescent gold nanoclusters which are protected by dihydrolipoic acid, mercaptoundecanoic acid and polyethylenimine. The fluorescent gold nanoclusters can perform a variety of bioconjugation processes such as PEGylation, biotinylation as well as forming complex nanobioconjugates with streptavidins. The brightening effects under proper surface modification are also reported. The clusters have a decent quantum yield, high colloidal stability, and can be readily conjugated with biological molecules. Nonspecific uptake by human aortic endothelial cells is demonstrated.

Effects of different PCR temperatures on primer conjugated quantum dots

The capability and effect of quantum dots on the detection of DNA concentration were investigated in this paper. The results showed that coating and conjugation procedure would not affect the electrical and optical properties of quantum dots. However, the optical property of quantum dots was altered under high and varied temperatures.