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Wen-Shuo Kuo,1,2,* Tien-Sung Yeh,3,* Chia-Yuan Chang,4 Jui-Chang Liu,2 Chang-Hsin Chen,5 Edmund Cheung So,6,7 Ping-Ching Wu8 1School of Chemistry and Materials Science, Nanjing University of Information Science and Technology, Nanjing 210044, Jiangsu, People’s Republic of China; 2Allergy & Clinical Immunology Research Center, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan, Republic of China; 3Department of Physical Medicine and Rehabilitation, An Nan Hospital, China Medical University, Tainan 709, Taiwan, Republic of China; 4Department of Mechanical Engineering, National Cheng Kung University, Tainan 701, Taiwan, Republic of China; 5AbVision Inc., Milpitas, CA 95035, USA; 6Department of Anesthesia & Medicine Research, An Nan Hospital, China Medical University, Tainan 709, Taiwan, Republic of China; 7Graduate Institute of Medical Sciences, Chang Jung Christian University, Tainan 711, Taiwan, Republic of China; 8Department of Biomedical Engineering, National Cheng Kung University, Tainan, 701, Taiwan, Republic of China*These authors contributed equally to this workCorrespondence: Edmund Cheung So; Ping-Ching Wu Email edmundsotw@gmail.com; wbcxyz@bme.ncku.edu.twBackground: Although graphene quantum dots (GQDs) have received considerable research attention for their applications in various fields, the use of GQDs, such as nitrogen-doped GQDs (N-GQDs) and amino-functionalized N-GQDs (amino-N-GQDs), as photosensitizers to facilitate photodynamic therapy (PDT) has received limited research intention. To address this research gap, this study prepared novel amino-N-GQDs and investigated their properties.Methods: The amino-N-GQDs subjected to two-photon excitation (TPE) exhibited remarkable bactericidal capability in PDT. The bonding compositions of nitrogen and the amino-functionalized group played a critical role in their antimicrobial effects.Results: Compared with amino-group-free N-GQDs and amino-N-free GQDs, the amino-N-GQDs generated a higher amount of reactive oxygen species, demonstrating their superior efficacy for two-photon PDT. Additionally, the intrinsic luminescence properties and high photostability of the amino-N-GQDs demonstrate their suitability as an effective two-photon contrast agent for tracking bacteria during two-photon biomedical imaging.Conclusion: The amino-N-GQD and their remarkable properties may provide an efficient alternative approach for observing and easily eliminating malignant microbes in the future.Keywords: nitrogen atom-doped graphene quantum dots, amino-functionalized and nitrogen atom-doped graphene quantum dots, two-photon photodynamic therapy, two-photon contrast agent, two-photon biomedical imaging

BackgroundNitrogen doping and amino-group functionalization, which result in strong electron donation, can be achieved through chemical modification. Large π-conjugated systems of graphene quantum dot (GQD)-based materials acting as electron donors can be chemically manipulated with low two-photon excitation energy in a short photoexcitation time for improving the charge transfer efficiency of sorted nitrogen-doped amino acid–functionalized GQDs (sorted amino-N-GQDs). ResultsIn this study, a self-developed femtosecond Ti-sapphire laser optical system (222.7 nJ pixel−1 with 100-170 scans, approximately 0.65-1.11 s of total effective exposure times; excitation wavelength: 960 nm in the near-infrared II region) was used for chemical modification. The sorted amino-N-GQDs exhibited enhanced two-photon absorption, post-two-photon excitation stability, two-photon excitation cross-section, and two-photon luminescence through the radiative pathway. The lifetime and quantum yield of the sorted amino-N-GQDs decreased and increased, respectively. Furthermore, the sorted amino-N-GQDs exhibited excitation-wavelength-independent photoluminescence in the near-infrared region and generated reactive oxygen species after two-photon excitation. An increase in the size of the sorted amino-N-GQDs boosted photochemical and electrochemical efficacy and resulted in high photoluminescence quantum yield and highly efficient two-photon photodynamic therapy. Conclusion The sorted dots can be used in two-photon contrast probes for tracking and localizing analytes during two-photon imaging in a biological environment and for conducting two-photon photodynamic therapy for eliminating infectious microbes.

In this study, sorted nitrogen-doped graphene quantum dots were prepared and subsequently conjugated with polymers. The synthesized materials exhibited excitation-wavelength-independent photoluminescence emissions ranging from ultraviolet to near-infrared and were 0.9-8.4 nm in size. The materials also exhibited high-photoluminescence quantum yields and excellent two-photon properties. Therefore, in two-photon bioimaging the materials with different emission spectra can be effective two-photon contrast agents. Specific antibodies were used to label organelles in cancer cells and identify nuclear antigens, thereby enabling the simultaneous detection of four targets in cells at a single two-photon excitation wavelength. The sorted nitrogen-doped graphene quantum dot materials were determined to be considerably more advantageous than organic dyes in identifying multiplexed targets, and they can be effective probes in cellular imaging.

We successfully prepared water-soluble fullerenol [C60(OH)46] that exhibited a high singlet oxygen quantum yield and efficiently generated reactive oxygen species. Additionally, the water-soluble C60(OH)46 with a higher composition of exposed hydroxyl groups had superior two-photon stability and characteristics compared with that with a lower composition of such groups. Therefore, the prepared fullerenol can be an effective two-photon photosensitizer. The water-soluble C60(OH)46 had favorable two-photon properties. During two-photon photodynamic therapy, the water-soluble C60(OH)46 had substantial antimicrobial activity against Escherichia coli at an ultralow-energy level of 211.2 nJ pixel−1 with 800 scans and a photoexcited wavelength of 760 nm.

We fabricated nanomaterials comprising amino-functionalized and nitrogen-doped graphene quantum dots (amino-N-GQDs) and investigated their photostability and intrinsic luminescence in the near-infrared spectrum to determine their suitability as contrast agents in two-photon imaging (TPI). We observed that amino-N-GQDs with a higher amount of bonded nitrogen and amino-functionalized groups (6.2%) exhibited superior two-photon properties to those with a lower amount of such nitrogen and groups (4.9%). These materials were conjugated with polymers containing sulfur (polystyrene sulfonate, PSS) and nitrogen atoms (polyethylenimine, PEI), forming amino-N-GQD&ndash, PSS&ndash, PEI specimens (amino-N-GQD-polymers). The polymers exhibited a high quantum yield, remarkable stability, and notable two-photon properties and generated no reactive oxygen species, rendering them excellent two-photon contrast agents for bioimaging. An antiepidermal growth factor receptor (AbEGFR) was used for labeling to increase specificity. Two-photon imaging (TPI) of amino-N-GQD (6.2%)-polymer-AbEGFR-treated A431 cancer cells revealed remarkable brightness, intensity, and signal-to-noise ratios for each observation at a two-photon excitation power of 16.9 nJ pixel&minus, 1 under 30 scans and a three-dimensional (3D) depth of 105 &micro, m, indicating that amino-N-GQD (6.2%)-polymer-AbEGFR-treated cells can achieve two-photon luminescence with 71 times less power required for two-photon autofluorescence (1322.8 nJ pixel&minus, 1 with 500 scans) of similar intensity. This economy can minimize photodamage to cells, rendering amino-N-GQD-polymers suitable for noninvasive 3D bioimaging.

A Sn-doped TiO2 nanocrystallite with the composition of Sn0.1Ti0.9O2 was prepared by hydrothermal co-precipitation followed by thermal annealing at 600 °C for 2 hours. Results combining high-resolution transmission electron microscopy, in situ XRD, ex situ XAS, and electrochemical impedance spectroscopy indicate that Sn0.1Ti0.9O2 underwent restructuring to give Sn-substituted TiO2 (Sn to Ti sites) by a lithiation reaction at 0.1C in a lithium-ion battery. The capacity and cyclability of Sn0.1Ti0.9O2 were increased by ∼73% (to ∼598 mA h g−1) and ∼17.4% compared with those of TiO2 (∼345 mA h g−1 and ∼22%) at 0.1C until the 250th cycle. Our results proved that these improvements were caused by the self-aligned formation of Sn–Ti oxide and the subsequent formation of a SnLix local alloy at Ti sites.

Nitrogen-doped graphene quantum dot (N-GQD) nanomaterials conjugated with polyethylenimine (PEI)-polystyrene sulfonate (PSS)-anti-epidermal growth factor receptor (AbEGFR) antibody (N-GQD-PEI-PSS-AbEGFR) demonstrated impressive two-photon properties and stability, signifying that they can serve as an effective two-photon contrast agent in two-photon bioimaging. Furthermore, they provided high intensity, brightness, and signal-to-noise ratios at an ultra-low two-photon excitation (TPE) power level in an observation extending to a deep, three-dimensional depth.

To develop a drug delivery system (DDS), it is critical to address challenging tasks such as the delivery of hydrophobic and amphiphilic compounds, cell uptake, and the metabolic fate of the drug delivery carrier. Low-density lipoprotein (LDL) has been acknowledged as the human serum transporter of natively abundant lipoparticles such as cholesterol, triacylglycerides, and lipids. Apolipoprotein B (apo B) is the only protein contained in LDL, and possesses a binding moiety for the LDL receptor that can be internalized and degraded naturally by the cell. Therefore, synthetic/reconstituting apoB lipoparticle (rABL) could be an excellent delivery carrier for hydrophobic or amphiphilic materials. Here, we synthesized rABL in vitro, using full-length apoB through a five-step solvent exchange method, and addressed its potential as a DDS. Our rABL exhibited good biocompatibility when evaluated with cytotoxicity and cell metabolic response assays, and was stable during storage in phosphate-buffered saline at 4 °C for several months. Furthermore, hydrophobic superparamagnetic iron oxide nanoparticles (SPIONPs) and the anticancer drug M4N (tetra-O-methyl nordihydroguaiaretic acid), used as an imaging enhancer and lipophilic drug model, respectively, were incorporated into the rABL, leading to the formation of SPIONPs- and M4N- containing rABL (SPIO@rABL and M4N@rABL, respectively). Fourier transform infrared spectroscopy suggested that rABL has a similar composition to that of LDL, and successfully incorporated SPIONPs or M4N. SPIO@rABL presented significant hepatic contrast enhancement in T2-weighted magnetic resonance imaging in BALB/c mice, suggesting its potential application as a medical imaging contrast agent. M4N@rABL could reduce the viability of the cancer cell line A549. Interestingly, we developed solution-phase high-resolution transmission electron microscopy to observe both LDL and SPIO@rABL in the liquid state. In summary, our LDL-based DDS, rABL, has significant potential as a novel DDS for hydrophobic and amphiphilic materials, with good cell internalization properties and metabolicity.

A graphene quantum dot (GQD) used as the photosensitizer with high two-photon absorption in the near-infrared region, a large absolute cross section of two-photon excitation (TPE), strong two-photon luminescence, and impressive two-photon stability could be used for dual modality two-photon photodynamic therapy (PDT) and two-photon bioimaging with an ultrashot pulse laser (or defined as TPE). In this study, a GQD efficiently generated reactive oxygen species coupled with TPE, which highly increased the effective PDT ability of both Gram-positive and -negative bacteria, with ultralow energy and an extremely short photoexcitation time generated by TPE. Because of its two-photon properties, a GQD could serve as a promising two-photon contrast agent for observing specimens in depth in three-dimensional biological environments while simultaneously proceeding with PDT action to eliminate bacteria, particularly in multidrug-resistant (MDR) strains. This procedure would provide an efficient alternative approach to easily cope with MDR bacteria.

This study reports a new microfluidic cell culture platform for real-time, in vitro microscopic observation and evaluation of cellular functions. Microheaters, a micro temperature sensor, and micropumps are integrated into the system to achieve a self-contained, perfusion-based, cell culture microenvironment. The key feature of the platform includes a unique, ultra-thin, culture chamber with a depth of 180 mum, allowing for real-time, high-resolution cellular imaging by combining bright field and fluorescent optics to visualize nanoparticle-cell/organelle interactions. The cell plating, culturing, harvesting and replenishing processes are performed automatically. The developed platform also enables drug screening and real-time, in situ investigation of the cellular and sub-cellular delivery process of nano vectors. The mitotic activity and the interaction between cells and the nano drug carriers (conjugated quantum dots-epirubicin) are successfully monitored in this device. This developed system could be a promising platform for a wide variety of applications such as high-throughput, cell-based studies and as a diagnostic cellular imaging system.

Yen-Chin Liu1, Ping-Ching Wu2, Dar-Bin Shieh2–5, Sheng-Nan Wu3,6,71Department of Anesthesiology, 2Institute of Oral Medicine and Department of Stomatology, 3Department of Physiology, National Cheng Kung University Hospital, College of Medicine, 4Advanced Optoelectronic Technology Center, 5Center for Micro/Nano Science and Technology, National Cheng Kung University, 6Innovation Center for Advanced Medical Device Technology, National Cheng Kung University, 7Department of Anatomy and Cell Biology, National Cheng Kung University Medical College, Tainan, TaiwanAims: Fe3O4 nanoparticles (NPs) have been known to provide a distinct image contrast effect for magnetic resonance imaging owing to their super paramagnetic properties on local magnetic fields. However, the possible effects of these NPs on membrane ion currents that concurrently induce local magnetic field perturbation remain unclear.Methods: We evaluated whether amine surface-modified Fe3O4 NPs have any effect on ion currents in pituitary tumor (GH3) cells via voltage clamp methods.Results: The addition of Fe3O4 NPs decreases the amplitude of membrane electroporation-induced currents (IMEP) with a half-maximal inhibitory concentration at 45 µg/mL. Fe3O4 NPs at a concentration of 3 mg/mL produced a biphasic response in the amplitude of IMEP, ie, an initial decrease followed by a sustained increase. A similar effect was also noted in RAW 264.7 macrophages.Conclusion: The modulation of magnetic electroporation-induced currents by Fe3O4 NPs constitutes an important approach for cell tracking under various imaging modalities or facilitated drug delivery.Keywords: iron oxide, ion current, free radical

Apart from the reported PLGA submicro- and microspheres with broad size distribution, we have successfully developed a methodology using nanoprecipitation to prepare different sizes of PLGA nanoparticles with narrow size distributions. The newly developed PLGA nanoparticles could be readily modified with hydrophilic biomaterials on their surface and entrap hydrophobic drugs into their interiors. The encapsulation of FITC inside PLGA nanoparticles displayed a controlled release of drug system. The surfaces of the FITC entrapped PLGA nanoparticles were conjugated with quantum dots to serve as bimodal imaging probes. For nuclear transport, combination of nuclear localization signal (NLS) and PLGA nanoparticles, PLGA nanoparticles could successfully enter into HeLa cells nuclei. From tissue uptake results, PLGA nanoparticles had more uptaken by brain and liver than other tissues. The iron oxide nanoparticles-conjugated PLGA nanoparticle showed high efficiency of relaxivities r2 and could be used as the powerful magnetic resonance imaging (MRI) agents.

The first in vivo optical virtual biopsy based on epi-third-harmonic-generation (THG) microscopy is successfully demonstrated using Syrian hamster oral mucosa as a model system. Without complex physical biopsy procedures, epi-THG microscopy can provide high spatial resolution dynamic images of oral mucosa and sub-mucosa in all three dimensions. The demonstrated intra-vital epi-THG microscopy provide high resolution observation of blood flow in the capillary and could be a promising tool to image angiogenesis, which is an important feature for many human diseases including malignancies. The system setup of epi-THG microscopy can be easily integrated with other nonlinear optical microscopy such as second-harmonic generation and multi-photon fluorescence microscopy by using the same laser system to provide better integrated molecular and structural information for future clinical diagnosis. By adding 6% acetic acid solution on the mucosa, THG contrast on the borders of nuclei was found to be greatly enhanced due to the alterations of their linear and nonlinear THG susceptibilities. With a virtual-transition-based technology without using fluorescence, the optical epi-THG biopsy we demonstrated shows promise for future noninvasive in vivo diseases examinations.