Walter H. Chang

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)

STUDIES OF PHOTOKILLING OF BACTERIA USING TITANIUM DIOXIDE NANOPARTICLES

Metal pins used to apply skeletal traction or external fixation devices protruding through skin are susceptible to the increased incidence of pin site infection. In this work, we tried to establish the photokilling effects of titanium dioxide (TiO2) nanoparticles on an orthopedic implant with an in vitro study. In these photocatalytic experiments, aqueous TiO2 was added to the tested microorganism. The time effect of TiO2 photoactivation was evaluated, and the loss of viability of five different bacteria suspensions (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus hirae, and Bacteroides fragilis) was examined by the viable count procedure. The bactericidal effect of TiO2 nanoparticle-coated metal plates was also tested. The ultraviolet (UV) dosage used in this experiment did not affect the viability of bacteria, and all bacteria survived well in the absence of TiO2 nanoparticles. The survival curve of microorganisms in the presence of TiO2 nanoparticles showed that nearly complete killing was achieved after 50 min of UV illumination. The formation of bacterial colonies above the TiO2 nanoparticle-coated metal plates also decreased significantly. In this study, we clearly demonstrated the bactericidal effects of titanium dioxide nanoparticles. In the presence of UV light, the titanium dioxide nanoparticles can be applicable to medical facilities where the potential for infection should be controlled.

Modulation of Osteogenesis in Human Mesenchymal Stem Cells by Specific Pulsed Electromagnetic Field Stimulation

Human mesenchymal stem cells (hMSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications by virtue of their capacity for self‐renewal and multipotent differentiation. Our intent was to characterize the effect of pulsed electromagnetic fields (PEMFs) on the proliferation and osteogenic differentiation of hMSCs in vitro. hMSCs isolated from the bone marrow of adult patients were cultured with osteogenic medium for up to 28 days and exposed to daily PEMF stimulation with single, narrow 300 µs quasi‐rectangular pulses with a repetition rate of 7.5 Hz. Relatively greater cell numbers were observed at late stages of osteogenic culture with PEMF exposure. The production of alkaline phosphatase (ALP), an early marker of osteogenesis, was significantly enhanced at day 7 with PEMF treatment in both basal and osteogenic cultures as compared to untreated controls. Furthermore, the expressions of other early osteogenic genes, including Runx2/Cbfa1 and ALP, were also partially modulated by PEMF exposure, indicating that osteogenesis in hMSCs was associated with the specific PEMF stimulation. Based on ALP and alizarin red S staining, the accumulation of ALP protein produced by the hMSCs as well as calcium deposits reached their highest levels at day 28. Our results indicate that extremely low‐frequency PEMF stimulation may play a modulating role in hMSC osteogenesis. Taken together, these findings provide insights on the development of PEMF as an effective technology for regenerative medicine.

Fluorescent Gold Nanoclusters as a Biocompatible Marker for In Vitro and In Vivo Tracking of Endothelial Cells

We have been investigating the fluorescent property and biocompatibility of novel fluorescent gold nanoclusters (FANC) in human aortic endothelial cells (HAEC) and endothelial progenitor cells (EPC). FANC (50-1000 nmol/L) was delivered into cells via the liposome complex. The fluorescence lasted for at least 28 days with a half-life of 9 days in vitro. Examination of 12 transcripts regulating the essential function of endothelial cells after a 72 h delivery showed that only the vascular cell adhesion molecule 1 and the vascular endothelial cadherin were down-regulated at high concentration (500 nmol/L). In addition, no activation of caspase 3 or proliferating cell nuclear antigens was detected. 3-[4,5-Dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) assay demonstrated that, unlike the markedly suppressed viability in cells treated with quantum dots, FANC had minimal effect on the viability, unless above 500 nmol/L, at which level a minor reduction of viability mainly caused by liposome was found. Tube formation assay showed no impaired angiogenesis in the EPC treated with FANC. In vivo study using hindlimb ischemic mice with an intramuscular injection of FANC-labeled human EPC showed that the cells preserved an angiogenic potential and exhibited traceable signals after 21 days. These findings demonstrated that FANC is a promising biocompatible fluorescent probe.

In vitro effects of low-intensity ultrasound stimulation on the bone cells

Mechanical perturbations serve as extracellular signals to a variety of cells, including bone cells. Low-intensity pulsed ultrasound produces significant multifunctional effects that are directly relevant to bone formation and resorption. Ultrasound stimulation has been shown to accelerate bone-defect healing and trabecular bone regeneration. In this study, we use an in vitro bone cell culture model to investigate the effect of low-intensity pulsed ultrasound. The rat alveolar mononuclear cell-calvaria osteoblast coculture system was used in this study. Before treatment, the bone cells were cultured for 3 days to facilitate their attachment and differentiation. Then, ultrasound exposure (frequency = 1 MHz, intensity = 0.068 W/cm(2)) or sham exposure for 20 min per day was applied until the end of the experiment. Half of the culture media were obtained on the 4th, 5th, 6th, 7th, 8th, 9th, and 10th days for the analysis of cytokines and biochemical parameters. At the end of the experiment, cells were fixed and stained for identification and quantification of the osteoblast and osteoclast cells. After low-intensity pulse ultrasound stimulation, the osteoblast cell counts were significantly increased, whereas the osteoclast cell counts were significantly decreased. The total alkaline phosphatase amount in the culture medium was increased after 7 days of ultrasound stimulation, and tumor necrosis factor-alpha in ultrasound-stimulated bone cells was significantly increased after the 7th day of culture and reached 474.77% of the control medium on the 10th day of culture. The results of this study suggest that low-intensity ultrasound treatment may have a stimulatory effect on bone-healing processes.

Bioanalytics and biolabeling with semiconductor nanoparticles (quantum dots)

In this mini-review recent applications of quantum dots in bioanalytics and biolabeling are discussed. The state-of-the-art of the field is summarized, some selected applications are highlighted, and future directions are suggested.

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.

Influence of hydroxyapatite particle size on bone cell activities: an in vitro study.

Over the past decade, a large number of biomaterials have been proposed as artificial bone fillers for repairing bone defects. The material most widely used in clinical medicine is hydroxyapatite. The aim of our investigation was to study the effect of hydroxyapatite size mechanism on osteoblasts. The osteoblasts were cultured in vitro with 0.1% (1 mg/mL) of various sized hydroxyapatite particles (0.5-3.0, 37-63, 177-250, and 420-841 microm) for 1 h, 3 h, 1 day, 3 days, and 7 days. The results showed that adding hydroxyapatite particles to osteoblast cultures can significantly affect osteoblast cell count. Osteoblast populations decreased significantly. Osteoblast mean surface areas also changed significantly. Transforming growth factor-beta1 (TGF-beta1) concentrations in culture medium decreased significantly with the addition of hydroxyapatite particles. Prostaglandin E2 (PGE2) concentrations in medium increased significantly. The changes in TGF-beta1 and PGE2 concentration were more significant and persisted longer in smaller-particle groups. The inhibitory effects of hydroxyapatite particles on osteoblast cell cultures were mediated by the increased synthesis of PGE2. Caution should be exercised before using a hydroxyapatite product which could easily break down into fine particles.

The influence of hydroxyapatite particles on osteoclast cell activities

Aseptic loosening after total joint arthroplasty is a major problem in orthopedic surgery. Small particles from material wear have been reported as the main cause of implant failure. For this reason, investigation into possible wear particles from the materials used in the implant may lead to longevity after arthroplasty. Hydroxyapatite (HA) has been extensively investigated and reported as an excellent biomaterial with excellent biocompatibility. In this study, we used an in vitro osteoblast/osteoclast model to test the biocompatibility of various-sized HA particles. Primary osteoclasts/osteoblasts were co-cultured with different-sized HA particles (0.5-3.0 microm, 37-53 microm, 177-205 microm, and 420-841 microm) for 3 h, 1 day, 3 days, and 7 days. Cellular responses to the HA particles were evaluated by changes in cell counts and the secretion of transforming growth factor (TGF-beta1), alkaline phosphatase (ALP), tumor necrosis factor (TNF-alpha), prostaglandin (PGE2), and lactate dehydrogenase (LDH) in the supernatant of the culture media. The results showed that osteoblasts/osteoclasts co-cultured with HA particles smaller than 53 microm undergo the most significant changes. Cellular counts significantly decreased, and the changes were more obvious in the osteoblast population. There also was a significant decrease in TGF-beta1 concentration and a significant increase in PGE2 and LDH concentration, but there were no changes in the TNF-alpha or ALP titer. It can be concluded that larger HA particles may be quite compatible with bone cells while smaller-sized HA particles can both activate the osteoclasts and decrease the cell population of the osteoblasts. Justification for the additional expense incurred with the use of hydroxyapatite in primary total hip arthroplasty should be further evaluated.