Surface charge-dependent cytokine production using near-infrared emitting silicon quantum dots.

Toll-like receptor 9 (TLR-9) is a protein that helps our immune system identify specific DNA types. Upon detection, CpG oligodeoxynucleotides signal the immune system to generate cytokines, essential proteins that contribute to the body's defence against infectious diseases. Native phosphodiester type B CpG ODNs induce only Interleukin-6 with no effect on interferon-α. We prepared silicon quantum dots containing different surface charges, such as positive, negative, and neutral, using amine, acrylate-modified Plouronic F-127, and Plouronic F-127. Then, class B CpG ODNs are loaded on the surface of the prepared SiQDs. The uptake of ODNs varies based on the surface charge; positively charged SiQDs demonstrate higher adsorption compared to SiQDs with negative and neutral surface charges. The level of cytokine production in peripheral blood mononuclear cells was found to be associated with the surface charge of SiQDs prior to the binding of the CpG ODNs. Significantly higher levels of IL-6 and IFN-α induction were observed compared to neutral and negatively charged SiQDs loaded with CpG ODNs. This observation strongly supports the notion that the surface charge of SiQDs effectively regulates cytokine induction.

Osteopontin Rejuvenates Senescent Adipose-Derived Stem Cells and Restores their Bone Tissue Regenerative Function.

The regenerative function of stem cells is compromised when the proportion of senescent stem cells increases with ageing advance. Therefore, combating stem cell senescence is of great importance for stem cell-based tissue engineering in the elderly, but remains largely unexplored. Osteopontin (OPN), a glycosylated phosphoprotein, is one of the key extracellular matrix molecules in bone tissue. OPN activates various signalling pathways and modulates cellular activities, including cell senescence. However, the role of OPN in stem cell senescence remains largely unknown. This study aims to investigate if OPN modulates cell senescence and bone regenerative function in human adipose-derived mesenchymal stem cells (ASCs), and to determine the underlying mechanisms. We first developed a senescent ASC model using serial passaging until passage 10 (P10), in which senescent cells were characterised by reduced proliferation and osteogenic differentiation capacity compared to P4 ASCs. The conditioned medium from P10 ASCs exhibited a diminished trophic effect on human osteoblasts (HOBs), compared to that from P4 ASCs. P10 ASCs on OPN-coated surface showed rejuvenated phenotype and enhanced osteogenic differentiation. The conditioned medium from P10 ASCs on OPN-coating improved trophic effects on HOBs. OPN regulated the morphology of senescent ASCs, transforming them from a more rounded and flattened cell shape to an elongated shape with a smaller area. These findings demonstrated the effects of OPN in restoring senescent ASCs functions, possibly through a mechanism that involves the modulation of cell morphology, indicating that OPN might hold a great potential for rejuvenating senescent stem cells and could potentially open a new venue for regenerating bone tissue in age-related diseases.

Ultrasensitive Dual Fluorophore-Conjugated Carbon Dots for Intracellular pH Sensing in 3D Tumor Models.

The dysregulation of pH has been linked to the onset of chronic conditions, such as cancer and neurological diseases. Consequently, the development of a highly sensitive tool for intracellular pH sensing is imperative to investigate the interplay between pH and the biochemical changes accompanying disease pathogenesis. Here, we present the development of a ratiometric fluorescent nanoprobe, NpRhoDot, designed for precisely measuring pH levels. We demonstrate its efficacy in sensitively reporting intracellular pH in monolayer A549 lung cancer cells, primary fibroblast cells, and 3D tumor spheroids derived from the DLD-1 colorectal adenocarcinoma cell line. NpRhoDot leverages a novel design, where stable carbon dots are functionalized with a pH-responsive ratiometric fluorescent probe comprising a naphthalimide-rhodamine moiety, NpRho1. This design confers NpRhoDot with the high pH sensitivity characteristics of organic fluorescent probes, along with excellent photostability up to 1 h and biocompatibility of carbon dots. Through one-photon and two-photon fluorescence microscopy, we validate the reliability of NpRhoDot for biosensing intracellular pH in monolayer and three-dimensional tumor models from pH 4 to 7.

Nicotinamide Mononucleotide Alleviates Osteoblast Senescence Induction and Promotes Bone Healing in Osteoporotic Mice.

Combating the accumulated senescent cells and the healing of osteoporotic bone fractures in the older remains a significant challenge. Nicotinamide mononucleotide (NMN), a precursor of NAD+, is an excellent candidate for mitigating aging-related disorders. However, it is unknown if NMN can alleviate senescent cell induction and enhance osteoporotic bone fracture healing. Here we show that NMN treatment partially reverses the effects of tumor necrosis factor-alpha (TNF-α) on human primary osteoblasts (HOBs): senescent cell induction, diminished osteogenic differentiation ability, and intracellular NAD+ and NADH levels. Mechanistically, NMN restores the mitochondrial dysfunction in HOBs induced by TNF-α evidenced by increased mitochondrial membrane potential and reduced reactive oxidative species and mitochondrial mass. NMN also increases mitophagy activity by down-regulating P62 expression and up-regulating light chain 3B-II protein expression. In addition, the cell senescence protective effects of NMN on HOBs are mitigated by a mitophagy inhibitor (Bafilomycin A1). In vivo, NMN supplementation attenuates senescent cell induction in growth plates, partially prevents osteoporosis in an ovariectomized mouse model, and accelerates bone healing in osteoporotic mice. We conclude that NMN can be a novel and promising therapeutic candidate to enhance bone fracture healing capacity in the older.

Promise and Perspective of Nanomaterials in Antisenescence Tissue Engineering Applications.

The tissue engineering approach for repair and regeneration has achieved significant progress over the past decades. However, challenges remain in developing strategies to solve the declined or impaired innate cell and tissue regeneration capacity that occurs with aging. Cellular senescence is a key mechanism underlying organismal aging and is responsible for the declined tissue regeneration capacity in the aging population. Therefore, to promote the diminished tissue regeneration ability in the aged population, it is critical to developing a feasible and promising strategy to target senescent cells. Recent advances in nanomaterials have revolutionized biomedical applications ranging from biosensing to bioimaging and targeted drug delivery. In this perspective, we review and discuss the nature and influences of cell-intrinsic and cell-extrinsic factors on reduced regenerative abilities through aging and how nanotechnology can be a therapeutic avenue to sense, rejuvenate, and eliminate senescent cells, thereby improving the tissue regeneration capacity in the aging population.

Influence of carbon dot synthetic parameters on photophysical and biological properties.

Recently, carbon dots (CDs) have been widely investigated for biological applications in imaging. One-step hydrothermal synthesis is considered to be one of the most promising methods for the synthesis of CDs, due to its simple and rapid manipulation, flexible selection of ingredients, environmentally friendly conditions, and low-cost. A number of synthetic and post-synthetic parameters, including solvent, heating time, dopant quantity, and particle size distribution, play a crucial role in controlling the size and surface structure of CDs, which ultimately have influence on their photophysical and biological behavior. Despite the crucial role of each of these parameters in defining the yield and nature of synthesized CDs, they have not previously been rigorously optimized, particularly with respect to desired biological applications. Herein, we report our comprehensive optimization of the parameters employed for the hydrothermal synthesis of CDs to gain a better understanding of the effect of these parameters on optical properties, cytotoxicity, and cellular uptake efficiency. Furthermore, this work will open up new pathways toward the design of CDs with physiochemical properties tailored for specific biomedical applications such as bioimaging.

Two-Photon Dual-Emissive Carbon Dot-Based Probe: Deep-Tissue Imaging and Ultrasensitive Sensing of Intracellular Ferric Ions.

Carbon dots (CDs)-based nanoparticles have been extensively explored for biological applications in sensing and bioimaging. However, the major translational barriers to CDs for imaging and sensing applications include synthetic strategies to obtain monodisperse CDs with tunable structural, electronic, and optical properties in order to achieve high-resolution deep-tissue imaging, intracellular detection, and sensing of metal ions with high sensitivity down to nanomolar levels. Herein, we report a novel strategy to synthesize and develop a multifunctional nitrogen-doped CDs probe of different sizes using a new combination of carbon and nitrogen sources. Our results show that the structural characteristics (i.e., the surface density of emissive traps and bandgaps levels) depend on the size of the CDs, which ultimately influences their optical properties. This work also demonstrates the development of a two-photon dual-emissive fluorescent multifunctional probes (3-FCDs) by conjugating fluorescein isothiocyanate on the surface of nitrogen-doped CDs. 3-FCDs show excellent near-infrared two-photon excitation ability, single-wavelength excitation, high photostability, biocompatibility, low cytotoxicity, and good cell permeability. Using two-photon fluorescence imaging, our multifunctional probe shows excellent deep-tissue high-resolution imaging capabilities with penetration depth up to 3000 and 280 µm in hydrogel scaffold and pigskin tissue, respectively. The designed probe exhibits ultrasensitivity and specificity toward Fe3+ ions with a remarkable detection limit of 2.21 nM using two-photon excitation. In addition, we also demonstrate the use of multifunctional CDs probe for ultrasensitive exogenous and real-time endogenous sensing of Fe3+ ions and imaging in live fibroblasts with rapid response times for intracellular ferric ion detection.

Two-Photon Active Boron Nitride Quantum Dots for Multiplexed Imaging, Intracellular Ferric Ion Biosensing, and pH Tracking in Living Cells.

Nanoparticles are key vehicles for targeted therapies because they can pass through biological barriers, enter into cells, and distribute within cell structures. We investigated the synthesis of blue and green emissive hexagonal boron nitride quantum dots (hBNQDs) using a liquid-exfoliation technique followed by hydrothermal treatment. A distinct shift from blue to bright-green emission was observed upon surface passivating the dots using poly(ethylene glycol) or PEG200 under the same UV irradiation. The quantum yield of the hBNQDs increased with the surface passivation. Multiplexed imaging was accomplished using the hBNQDs in conjunction with organic dyes. The hBNQDs provided images with distinctive emission wavelengths and fluorescence lifetimes. Although the fluorescence signals of blue- and green-emissive hBNQDs overlap spectrally with those of the emission wavelengths of the organic dyes, the fluorescence lifetime data were resolved temporally using software-based time gates. The blue-emissive hBNQD-b quantum dots were validated as sensitive platforms for detecting intracellular ferric ions with a low limit of detection (20.6 nM). The green-emissive hBNQD-g quantum dots successfully identified intracellular variations in pH, and the localization in human breast cancer cells was determined during their life cycles via fluorescence lifetime imaging.