A Non-Invasive Method for Monitoring Osteogenesis and Osseointegration Using Near-Infrared Fluorescent Imaging: A Model of Maxilla Implantation in Rats.

Currently, computed tomography and conventional X-ray radiography usually generate a micro-artifact around metal implants. This metal artifact frequently causes false positive or negative diagnoses of bone maturation or pathological peri-implantitis around implants. In an attempt to repair the artifacts, a highly specific nanoprobe, an osteogenic biomarker, and nano-Au-Pamidronate were designed to monitor the osteogenesis. In total, 12 Sprague Dawley rats were included in the study and could be chategorized in 3 groups: 4 rats in the X-ray and CT group, 4 rats in the NIRF group, and 4 rats in the sham group. A titanium alloy screw was implanted in the anterior hard palate. The X-ray, CT, and NIRF images were taken 28 days after implantation. The X-ray showed that the tissue surrounded the implant tightly; however, a gap of metal artifacts was noted around the interface between dental implants and palatal bone. Compared to the CT image, a fluorescence image was noted around the implant site in the NIRF group. Furthermore, the histological implant-bone tissue also exhibited a significant NIRF signal. In conclusion, this novel NIRF molecular imaging system precisely identifies the image loss caused by metal artifacts and can be applied to monitoring bone maturation around orthopedic implants. In addition, by observing the new bone formation, a new principle and timetable for an implant osseointegrated with bone can be established and a new type of implant fixture or surface treatment can be evaluated using this system.

Dihydrolipoic acid-coated gold nanocluster bioactivity against senescence and inflammation through the mitochondria-mediated JNK/AP-1 pathway.

Cellular senescence is the progressive impairment of function and proliferation in response to various regulators. Dihydrolipoic acid-coated gold nanoclusters (DHLA-Au NCs), which are molecular clusters with covalently linked dihydroxyl lipoic acid, preserve cellular activities for long-term incubation. DHLA-Au NC delivery was characterized, and we determined the role of growth supplements on internalization, allowing the optimization of DHLA-Au NC bioactivity. In the optimized medium, DHLA-Au NCs attenuated the levels of the senescence-associated phenotype. Molecular mechanism analysis further indicated that during DHLA-Au NC treatment, the activation of the stress signal JNK and its downstream c-Jun were impaired under LPS induction, which led to a decline in AP-1-mediated TNF-α transactivation. Confocal microscopy and subcellular fractionation analysis suggested that DHLA-Au NCs interacted with mitochondria through their lipid moiety and attenuated mitochondria-derived reactive oxygen species. With adequate treatment, DHLA-Au NCs show protection against cellular senescence and inflammation in vitro and in vivo.

Impact of dihydrolipoic acid on mouse embryonic stem cells and related regulatory mechanisms.

α-Lipoic acid (LA) is a thiol with antioxidant properties that protects against oxidative stress-induced apoptosis. LA is absorbed from the diet, taken up by cells and tissues, and subsequently reduced to dihydrolipoic acid (DHLA). Recently, DHLA has been used as the hydrophilic nanomaterial preparations, and therefore, determination of its bio-safety profile is essential. In this article, we show that DHLA (50-100 µM) induces apoptotic processes in mouse embryonic stem cells (ESC-B5), but exerts no injury effects at treatment dosages below 50 µM. Higher concentrations of DHLA (50-100 µM) directly increased the reactive oxygen species (ROS) content in ESC-B5 cells, along with a significant increase in cytoplasmic free calcium and nitric oxide (NO) levels, loss of mitochondrial membrane potential (MMP), activation of caspases-9 and -3, and cell death. Pretreatment with NO scavengers suppressed the apoptotic biochemical changes induced by 100 µM DHLA and promoted the gene expression levels of p53 and p21 involved in apoptotic signaling. Our results collectively indicate that DHLA at concentrations of 50-100 µM triggers apoptosis of ESC-B5 cells, which involves both ROS and NO. Importantly, at doses of less than 50 µM (0-25 µM), DHLA does not exert hazardous effects on ESC-B5 cell properties, including viability, development and differentiation. These results provide important information in terms of dosage safety and biocompatibility of DHLA to facilitate its further use as a precursor for biomaterial preparation.

Dihydrolipoic acid induces cytotoxicity in mouse blastocysts through apoptosis processes.

α-Lipoic acid (LA) is a thiol with antioxidant properties that protects against oxidative stress-induced apoptosis. LA is absorbed from the diet, taken up by cells and tissues, and subsequently reduced to dihydrolipoic acid (DHLA). In view of the recent application of DHLA as a hydrophilic nanomaterial preparation, determination of its biosafety profile is essential. In the current study, we examined the cytotoxic effects of DHLA on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, in vivo implantation by embryo transfer, and early embryonic development in an animal model. Blastocysts treated with 50 µM DHLA exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with DHLA were lower than that of their control counterparts. Moreover, in vitro treatment with 50 µM DHLA was associated with increased resorption of post-implantation embryos and decreased fetal weight. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 100 µM DHLA led to decreased early embryo development, specifically, inhibition of development to the blastocyst stage. However, it appears that concentrations of DHLA lower than 50 µM do not exert a hazardous effect on embryonic development. Our results collectively indicate that in vitro and in vivo exposure to concentrations of DHLA higher than 50 µM DHLA induces apoptosis and retards early pre- and post-implantation development, and support the potential of DHLA to induce embryonic cytotoxicity.

Integrating the microneedles with carboplatin to facilitate the therapeutic effect of radiotherapy for skin cancers.

In most skin cancer patients, excisional surgery is required to remove tumorous tissue. However, the risk of locoregional recurrence after surgery alone is relatively high, particularly for a locally advanced stage of melanoma. Therefore, additional adjuvant treatments, such as radiotherapy, can be used after surgery to inhibit recurrent melanoma after surgical removal. To enhance local radiotherapy, we present the combined X-ray radiation and radiosensitizers (carboplatin) through microneedles (MNs) to treat melanoma. The MNs could be beneficial to precisely delivering carboplatin into the sub-epidermal layer of the melanoma region and alleviate patients' fear and discomfort during the drug administration compared to the traditional local injection. The carboplatin was loaded into the tips of dissolving gelatin MNs (carboplatin-MNs) through the molding method. The results show gelatin MNs have sufficient mechanical strength and can successfully administer carboplatin into the skin. Both in vitro and in vivo studies suggest that carboplatin can enhance radiotherapy in melanoma treatment. With a combination of radiotherapy and carboplatin, the inhibition effect of carboplatin delivered into the B16F10 murine melanoma model through MNs administration (1.2 mg/kg) is equivalent to that through an intravenous route (5 mg/kg). The results demonstrate a promise of combined carboplatin and X-ray radiation treatment in treating melanoma by MNs administration.

Hemostasis and Anti-Inflammatory Abilities of AuNPs-Coated Chitosan Dressing for Burn Wounds.

Burn injuries are a common hazard in the military, as fire is likely to be weaponized. Thus, it is important to find an effective substance to accelerate burn wound healing. This study used chitosan and gold nanoparticles (AuNPs) as wound dressings and investigated their effectiveness in femoral artery hemorrhage swine and rat burn models. Chitosan dressing has significant hemostatic properties compared with gauze. Histological results showed that burn wounds treated with chitosan or AuNP-coated chitosan dressings exhibited more cells and a continuous structure of the epidermis and dermis than those of the control and untreated lesion groups. Furthermore, both chitosan dressings have been shown to positively regulate the expression of genes- and cytokines/chemokines-related to the wound healing process; AuNP-coated chitosan significantly lessened severe sepsis and inflammation, balanced the activities of pro-fibrotic and anti-fibrotic ligands for tissue homeostasis, regulated angiogenesis, and inhibited apoptosis activity, thereby being beneficial for the burn microenvironment. Hence, chitosan alone or in combination with AuNPs represents a prospective therapeutic substance as a burn dressing which might be helpful for burn wound care. This study provides a novel hemostasis dressing for modern warfare that is simple to use by most medical and paramedical personnel handling for burn treatment.

A Perspective on Imiquimod Microneedles for Treating Warts.

Warts are a common skin problem and are caused by infection with a virus. Warts are currently mainly treated by therapies involving ablating tissue or interrupting cellular division. However, all these existing treatments are either invasive or cause skin pain and tissue destruction. Imiquimod is a synthetic compound that belongs to the imidazoquinolinone family. It has been successfully used as a topical drug to treat external anogenital warts. However, topical imiquimod cream for warts is restricted by low skin permeability, and several side effects such as itching, pain, and erosions occur most frequently following topical treatment. Microneedle technology, a minimally invasive drug delivery system, has the potential to overcome the barrier of the stratum corneum. This technique would also offer a painless treatment choice and provide personalized therapies. In the study, we loaded imiquimod within dissolving microneedles using the molding method. Gelatin was used as a structural material for microneedle formation without adding a crosslinker. To our knowledge, this is the first study of using dissolving microneedles and exploring their utilization with imiquimod for the treatment of warts. First, we added fluorescent dye and trypan blue into the microneedles to evaluate the status of drugs in the microneedles and the degradation property of microneedles made of gelatin, respectively. Here we also prove the strength of the imiquimod microneedles and study their capability to penetrate the skin. The results show no apparent differences in mechanical failure after an additional imiquimod-loaded. Besides, we provide evidence that imiquimod microneedles induce secreted embryonic alkaline phosphatase (SEAP) in the RAW 264.7 macrophages. Gelatin does not affect the imiquimod in microneedles; a similar immune response was affected by the imiquimod alone or imiquimod complexed with gelatin. Our research demonstrates a proof of concept of using imiquimod microneedles for future warts treatment.

From mono-PEGylation towards anti-nonspecific protein interaction: comparison of dihydrolipoic acid versus glutathione-capped fluorescent gold nanoclusters using gel electrophoresis.

Ultrafine fluorescent gold nanoclusters (AuNCs) have emerged as biocompatible nanoprobes for biomedical imaging in vivo, and the precision surface chemistry of AuNCs is the key for attaining their clinical application. Comparison of two promising candidates for future nanomedicine, i.e. dihydrolipoic acid- versus glutathione-capped AuNCs (AuNC@DHLA vs. AuNC@GSH), was conducted for the first time to clarify their polyethylene glycol-related bioconjugate chemistry (PEGylation) and protein interactions. Gel electrophoresis was performed to separate the number of AuNCs PEGylation, and the molecular weight of the PEG spacer dominated the resolution of the separation in the gel. We have engineered and isolated the mono-PEGylated AuNCs either from the indirect carbodiimide bioconjugate chemistry or the direct Au-S binding. One-pot synthesis showed great efficiency for isolating mono-PEGylated AuNC@GSH from the tailored controlled aggregation of Au(i)-thiolate complexes on in situ generated Au(0) cores. Post-PEGylation of AuNC@GSH was also feasible using monodendate thiol-terminated PEG, but bidendate ligands of AuNC@DHLA exhibited low PEGylated efficiency by Au-S binding. In addition, mono-PEGylated AuNC@GSH significantly enhanced the ability of anti-nonspecific protein adsorption, but mono-PEGylated AuNC@DHLA cannot avoid the nonspecific binding with serum albumin. In addition, specific nano-assembly involving mono-biotinylated AuNCs with streptavidin were also compared using gel electrophoresis. These results provide key insights into the selection, preparation and design of functional AuNCs as nanoprobes for versatile biomedical applications.

Two new, near-infrared, fluorescent probes as potential tools for imaging bone repair.

A precise imaging technique to evaluate osteogenesis, osteodifferentiation, and osseointegration following peri-implant surgery is in high clinical demand. Herein, we report the generation of two new, near-infrared (NIR) fluorescent probes for use in the molecular imaging of bone repair. The first probe aims to monitor the in vitro differentiation of human mesenchymal stem cells (MSCs) into osteoblasts. A NIR fluorochrome was conjugated to a cyclic peptide that binds to integrin α5ß1, a factor that promotes osteogenesis in MSCs and therefore functioned as an osteoblast-specific marker. The second probe aims to monitor osteogenesis, and was generated by conjugating the drug pamidronate to a NIR fluorescent gold nanocluster. Pamidronate specifically binds to hydroxyapatite (HA), a mineral present in bone that is produced by osteoblasts, and therefore provides a functional marker for new bone formation. Our results show that both probes bind to their specific targets in vitro-differentiated osteoblasts, and not to undifferentiated MSCs, and emit NIR fluorescence for functional detection. This in vitro work demonstrates the ability of these probes to bind to active osteoblasts and their mineral deposits and highlight their potential utility as clinical tools for the imaging of the osseointegration process at the molecular level.

Tracking of cellular uptake of hydrophilic CdSe/ZnS quantum dots/hydroxyapatite composites nanoparticles in MC3T3-E1 osteoblast cells.

We report the fluorescent labeling of osteoblast cells using the biocompatible hydroxyapatite (HA) grown with nucleating seed of hydrophilic CdSe/ZnS quantum dots (QDs) allowing the real-time observation of cell under confocal microscope. We found that the MC3T3-E1 osteoblast cells can engulf HA with surface-tailored QDs showing fluorescent spots in the cytoplasm, while HA and QDs nanoparticles were not engulfed. It is interesting to see that the fluorescence was only displayed in the cytoplasm of MC3T3-E1 osteoblast cells. It can be envisioned that the nano-sized hydroxyapatite bearing fluorescent QD can only be internalized in the cytoplasm. Therefore, it is worth utilizing these composite particles to observe cellular physiology with minimal toxicity to the osteoblast cells.

Publisher Correction: Non-Toxic Gold Nanoclusters for Solution-Processed White Light-Emitting Diodes.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Non-Toxic Gold Nanoclusters for Solution-Processed White Light-Emitting Diodes.

Solution-processed optoelectronic devices are attractive because of the potential low-cost fabrication and the compatibility with flexible substrate. However, the utilization of toxic elements such as lead and cadmium in current optoelectronic devices on the basis of colloidal quantum dots raises environmental concerns. Here we demonstrate that white-light-emitting diodes can be achieved by utilizing non-toxic and environment-friendly gold nanoclusters. Yellow-light-emitting gold nanoclusters were synthesized and capped with trioctylphosphine. These gold nanoclusters were then blended with the blue-light-emitting organic host materials to form the emissive layer. A current efficiency of 0.13 cd/A was achieved. The Commission Internationale de l'Eclairage chromaticity coordinates of (0.27, 0.33) were obtained from our experimental analysis, which is quite close to the ideal pure white emission coordinates (0.33, 0.33). Potential applications include innovative lighting devices and monitor backlight.

Enhanced Conversion Efficiency of III-V Triple-junction Solar Cells with Graphene Quantum Dots.

Graphene has been used to synthesize graphene quantum dots (GQDs) via pulsed laser ablation. By depositing the synthesized GQDs on the surface of InGaP/InGaAs/Ge triple-junction solar cells, the short-circuit current, fill factor, and conversion efficiency were enhanced remarkably. As the GQD concentration is increased, the conversion efficiency in the solar cell increases accordingly. A conversion efficiency of 33.2% for InGaP/InGaAs/Ge triple-junction solar cells has been achieved at the GQD concentration of 1.2 mg/ml, corresponding to a 35% enhancement compared to the cell without GQDs. On the basis of time-resolved photoluminescence, external quantum efficiency, and work-function measurements, we suggest that the efficiency enhancement in the InGaP/InGaAs/Ge triple-junction solar cells is primarily caused by the carrier injection from GQDs to the InGaP top subcell.

Rapid transformation of protein-caged nanomaterials into microbubbles as bimodal imaging agents.

We present a general method for converting colloidal nanomaterials into microbubbles as ultrasound contrast agents. Protein-caged nanomaterials, made either by self-assembled nanoparticles' protein corona or by fluorescent gold nanoclusters, can be rapidly transformed into microbubbles via a sonochemical route, which promote disulfide cross-linking of cysteine residues between protein-caged nanomaterials and free albumin during acoustic cavitation. The proposed methods yielded microbubbles with multiple functions by adjusting the original nanoparticle/protein mixture. We also showed a new dual-modal imaging agent of fluorescent gold microbubbles in vitro and in vivo, which can hold many potential applications in medical diagnostics and therapy.

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.

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.