Targeting macrophages with multifunctional nanoparticles to detect and prevent atherosclerotic cardiovascular disease.

Despite the emergence of novel diagnostic, pharmacological, interventional, and prevention strategies, atherosclerotic cardiovascular disease remains a significant cause of morbidity and mortality. Nanoparticle (NP)-based platforms encompass diverse imaging, delivery, and pharmacological properties that provide novel opportunities for refining diagnostic and therapeutic interventions for atherosclerosis at the cellular and molecular levels. Macrophages play a critical role in atherosclerosis and therefore represent an important disease-related diagnostic and therapeutic target, especially given their inherent ability for passive and active NP uptake. In this review, we discuss an array of inorganic, carbon-based, and lipid-based NPs that provide magnetic, radiographic, and fluorescent imaging capabilities for a range of highly promising research and clinical applications in atherosclerosis. We discuss the design of NPs that target a range of macrophage-related functions such as lipoprotein oxidation, cholesterol efflux, vascular inflammation, and defective efferocytosis. We also provide examples of NP systems that were developed for other pathologies such as cancer and highlight their potential for repurposing in cardiovascular disease. Finally, we discuss the current state of play and the future of theranostic NPs. Whilst this is not without its challenges, the array of multifunctional capabilities that are possible in NP design ensures they will be part of the next frontier of exciting new therapies that simultaneously improve the accuracy of plaque diagnosis and more effectively reduce atherosclerosis with limited side effects.

A Telecom O-Band Emitter in Diamond.

Color centers in diamond are promising platforms for quantum technologies. Most color centers in diamond discovered thus far emit in the visible or near-infrared wavelength range, which are incompatible with long-distance fiber communication and unfavorable for imaging in biological tissues. Here, we report the experimental observation of a new color center that emits in the telecom O-band, which we observe in silicon-doped bulk single crystal diamonds and microdiamonds. Combining absorption and photoluminescence measurements, we identify a zero-phonon line at 1221 nm and phonon replicas separated by 42 meV. Using transient absorption spectroscopy, we measure an excited state lifetime of around 270 ps and observe a long-lived baseline that may arise from intersystem crossing to another spin manifold.

Photon extraction enhancement of praseodymium ions in gallium nitride nanopillars.

Lanthanoid-doped Gallium Nitride (GaN) integrated into nanophotonic technologies is a promising candidate for room-temperature quantum photon sources for quantum technology applications. We manufactured praseodymium (Pr)-doped GaN nanopillars of varying size, and showed significantly enhanced room-temperature photon extraction efficiency compared to unstructured Pr-doped GaN. Implanted Pr ions in GaN show two main emission peaks at 650.3 nm and 651.8 nm which are attributed to 3P0-3F2 transition in the 4f-shell. The maximum observed enhancement ratio was 23.5 for 200 nm diameter circular pillars, which can be divided into the emitted photon extraction enhancement by a factor of 4.5 and the photon collection enhancement by a factor of 5.2. The enhancement mechanism is explained by the eigenmode resonance inside the nanopillar. Our study provides a pathway for Lanthanoid-doped GaN nano/micro-scale photon emitters and quantum technology applications.

Targeting cell surface glycans with lectin-coated fluorescent nanodiamonds.

Glycosylation is arguably the most important functional post-translational modification in brain cells and abnormal cell surface glycan expression has been associated with neurological diseases and brain cancers. In this study we developed a novel method for uptake of fluorescent nanodiamonds (FND), carbon-based nanoparticles with low toxicity and easily modifiable surfaces, into brain cell subtypes by targeting their glycan receptors with carbohydrate-binding lectins. Lectins facilitated uptake of 120 nm FND with nitrogen-vacancy centers in three types of brain cells - U87-MG astrocytes, PC12 neurons and BV-2 microglia cells. The nanodiamond/lectin complexes used in this study target glycans that have been described to be altered in brain diseases including sialic acid glycans via wheat (Triticum aestivum) germ agglutinin (WGA), high mannose glycans via tomato (Lycopersicon esculentum) lectin (TL) and core fucosylated glycans via Aleuria aurantia lectin (AAL). The lectin conjugated nanodiamonds were taken up differently by the various brain cell types with fucose binding AAL/FNDs taken up preferentially by glioblastoma phenotype astrocyte cells (U87-MG), sialic acid binding WGA/FNDs by neuronal phenotype cells (PC12) and high mannose binding TL/FNDs by microglial cells (BV-2). With increasing recognition of glycans having a role in many diseases, the lectin bioconjugated nanodiamonds developed here are well suited for further investigation into theranostic applications.

Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device.

PURPOSE: Vitrification permits long-term banking of oocytes and embryos. It is a technically challenging procedure requiring direct handling and movement of cells between potentially cytotoxic cryoprotectant solutions. Variation in adherence to timing, and ability to trace cells during the procedure, affects survival post-warming. We hypothesized that minimizing direct handling will simplify the procedure and improve traceability. To address this, we present a novel photopolymerized device that houses the sample during vitrification. METHODS: The fabricated device consisted of two components: the Pod and Garage. Single mouse oocytes or embryos were housed in a Pod, with multiple Pods docked into a Garage. The suitability of the device for cryogenic application was assessed by repeated vitrification and warming cycles. Oocytes or early blastocyst-stage embryos were vitrified either using standard practice or within Pods and a Garage and compared to non-vitrified control groups. Post-warming, we assessed survival rate, oocyte developmental potential (fertilization and subsequent development) and metabolism (autofluorescence). RESULTS: Vitrification within the device occurred within ~ 3 nL of cryoprotectant: this volume being ~ 1000-fold lower than standard vitrification. Compared to standard practice, vitrification and warming within our device showed no differences in viability, developmental competency, or metabolism for oocytes and embryos. The device housed the sample during processing, which improved traceability and minimized handling. Interestingly, vitrification-warming itself, altered oocyte and embryo metabolism. CONCLUSION: The Pod and Garage system minimized the volume of cryoprotectant at vitrification-by ~ 1000-fold-improved traceability and reduced direct handling of the sample. This is a major step in simplifying the procedure.

Magnetic-field-dependent stimulated emission from nitrogen-vacancy centers in diamond.

Negatively charged nitrogen-vacancy (NV) centers in diamond are promising magnetic field quantum sensors. Laser threshold magnetometry theory predicts improved NV center ensemble sensitivity via increased signal strength and magnetic field contrast. Here, we experimentally demonstrate laser threshold magnetometry. We use a macroscopic high-finesse laser cavity containing a highly NV-doped and low absorbing diamond gain medium that is pumped at 532 nm and resonantly seeded at 710 nm. This enables a 64% signal power amplification by stimulated emission. We test the magnetic field dependency of the amplification and thus demonstrate magnetic field-dependent stimulated emission from an NV center ensemble. This emission shows an ultrahigh contrast of 33% and a maximum output power in the milliwatt regime. The coherent readout of NV centers pave the way for novel cavity and laser applications of quantum defects and diamond NV magnetic field sensors with substantially improved sensitivity for the health, research, and mining sectors.

Fabrication on the microscale: a two-photon polymerized device for oocyte microinjection.

PURPOSE: Intracytoplasmic sperm injection (ICSI) addresses male sub-fertility by injecting a spermatozoon into the oocyte. This challenging procedure requires the use of dual micromanipulators, with success influenced by inter-operator expertise. We hypothesized that minimizing oocyte handling during ICSI will simplify the procedure. To address this, we designed and fabricated a micrometer scale device that houses the oocyte and requires only one micromanipulator for microinjection. METHODS: The device consisted of 2 components, each of sub-cubic millimeter volume: a Pod and a Garage. These were fabricated using 2-photon polymerization. Toxicity was evaluated by culturing single-mouse presumptive zygotes (PZs) to the blastocyst stage within a Pod, with several Pods (and embryos) docked in a Garage. The development was compared to standard culture. The level of DNA damage/repair in resultant blastocysts was quantified (γH2A.X immunohistochemistry). To demonstrate the capability to carry out ICSI within the device, PZs were microinjected with 4-µm fluorescent microspheres and cultured to the blastocyst stage. Finally, the device was assessed for oocyte traceability and high-throughput microinjection capabilities and compared to standard microinjection practice using key parameters (pipette setup, holding then injecting oocytes). RESULTS: Compared to standard culture, embryo culture within Pods and a Garage showed no differences in development to the blastocyst stage or levels of DNA damage in resultant blastocysts. Furthermore, microinjection within our device removes the need for a holding pipette, improves traceability, and facilitates high-throughput microinjection. CONCLUSION: This novel device could improve embryo production following ICSI by simplifying the procedure and thus decreasing inter-operator variability.

Acoustomicrofluidic Concentration and Signal Enhancement of Fluorescent Nanodiamond Sensors.

Diamond nitrogen-vacancy (NV) centers constitute a promising class of quantum nanosensors owing to the unique magneto-optic properties associated with their spin states. The large surface area and photostability of diamond nanoparticles, together with their relatively low synthesis costs, make them a suitable platform for the detection of biologically relevant quantities such as paramagnetic ions and molecules in solution. Nevertheless, their sensing performance in solution is often hampered by poor signal-to-noise ratios and long acquisition times due to distribution inhomogeneities throughout the analyte sample. By concentrating the diamond nanoparticles through an intense microcentrifugation effect in an acoustomicrofluidic device, we show that the resultant dense NV ensembles within the diamond nanoparticles give rise to an order-of-magnitude improvement in the measured acquisition time. The ability to concentrate nanoparticles under surface acoustic wave (SAW) microcentrifugation in a sessile droplet is, in itself, surprising given the well-documented challenge of achieving such an effect for particles below 1 µm in dimension. In addition to a demonstration of their sensing performance, we thus reveal in this work that the reason why the diamond nanoparticles readily concentrate under the SAW-driven recirculatory flow can be attributed to their considerably higher density and hence larger acoustic contrast compared to those for typical particles and cells for which the SAW microcentrifugation flow has been shown to date.

Preferential coupling of diamond NV centres in step-index fibres.

Diamonds containing the negatively charged nitrogen-vacancy centre are a promising system for room-temperature magnetometry. The combination of nano- and micro-diamond particles with optical fibres provides an option for deploying nitrogen-vacancy magnetometers in harsh and challenging environments. Here we numerically explore the coupling efficiency from nitrogen-vacancy centres within a diamond doped at the core/clad interface across a range of commercially available fibre types so as to inform the design process for a diamond in fibre magnetometers. We determine coupling efficiencies from nitrogen-vacancy centres to the guided modes of a step-index fibre and predict the optically detected magnetic resonance (ODMR) generated by a ensemble of four nitrogen-vacancy centres in this hybrid fibre system. Our results show that the coupling efficiency is enhanced with a high refractive index difference between the fibre core and cladding and depends on the radial position of the nitrogen-vacancy centres in the fibre core. Our ODMR simulations show that due to the preferential coupling of the nitrogen-vacancy emission to the fibre guided modes, certain magnetometry features such as ODMR contrast can be enhanced and lead to improved sensitivity in such diamond-fibre systems, relative to conventional diamond only ensemble geometries.

An advanced method for quantitative measurements of cholesterol crystallization.

BACKGROUND: Cholesterol crystallization within an atherosclerotic plaque significantly contributes to the acceleration of plaque rupture - a problematic event due to the current lack of specific treatments to prevent such formations. Modelling this pathogenic process is also difficult due to the lack of suitable experimental models that enable quantitative analysis of crystal formation and bioactivity screening of potential therapeutic compounds. AIM: To develop an in vitro human cell model of cholesterol crystallization combined with an imaging system that incorporates both quantitative analysis and real-time continuous imaging of cholesterol crystal formation. METHODS AND RESULTS: An enhanced in vitro model of cholesterol crystallization was developed through the use of acetylated low-density lipoprotein (AcLDL) and 7-ketocholesterol as agents of foam cell induction within a human THP-1 monocytic cell line. Advanced confocal and polarizing microscopies were incorporated into the model so as to allow for quantitation of cholesterol crystallization, with the lipid-loaded group producing significantly greater numbers of cholesterol crystals than the untreated group. The utility of this system was also demonstrated by investigating the effects of the cholesterol-lowering drug lovastatin and therapeutic bile compound ursodeoxycholic acid (UDCA), showing that these drugs influence different aspects of cholesterol crystal formation. CONCLUSIONS: The in vitro human THP-1 monocyte model of cholesterol crystallization provides an effective and efficient means of quantitating cholesterol crystallization in the pre-clinical stage of research. The model also allows for the screening of potentially therapeutic compounds that may be used in attenuating or preventing cholesterol crystallization.

Multimodal Imaging and Soft X-Ray Tomography of Fluorescent Nanodiamonds in Cancer Cells.

Multimodal imaging promises to revolutionize the understanding of biological processes across scales in space and time by combining the strengths of multiple imaging techniques. Fluorescent nanodiamonds (FNDs) are biocompatible, chemically inert, provide high contrast in light- and electron-based microscopy, and are versatile optical quantum sensors. Here it is demonstrated that FNDs also provide high absorption contrast in nanoscale 3D soft X-ray tomograms with a resolution of 28 nm in all dimensions. Confocal fluorescence, atomic force, and scanning electron microscopy images of FNDs inside and on the surface of PC3 cancer cells with sub-micrometer precision are correlated. FNDs are found inside ≈1 µm sized vesicles present in the cytoplasm, providing direct evidence of the active uptake of bare FNDs by cancer cells. Imaging artefacts are quantified and separated from changes in cell morphology caused by sample preparation. These results demonstrate the utility of FNDs in multimodal imaging, contribute to the understanding of the fate of FNDs in cells, and open up new possibilities for biological imaging and sensing across the nano- and microscale.

Electrospun Nanodiamond-Silk Fibroin Membranes: A Multifunctional Platform for Biosensing and Wound-Healing Applications.

Next generation wound care technology capable of diagnosing wound parameters, promoting healthy cell growth, and reducing pathogenic infections noninvasively would provide patients with an improved standard of care and accelerated wound repair. Temperature is one of the indicating biomarkers specific to chronic wounds. This work reports a hybrid, multifunctional optical material platform-nanodiamond (ND)-silk membranes as biopolymer dressings capable of temperature sensing and promoting wound healing. The hybrid structure was fabricated through electrospinning, and 3D submicron fibrous membranes with high porosity were formed. Silk fibers are capable of compensating for the lack of an extracellular matrix at the wound site, supporting the wound-healing process. Negatively charged nitrogen vacancy (NV-) color centers in NDs exhibit optically detected magnetic resonance (ODMR) and act as nanoscale thermometers. This can be exploited to sense temperature variations associated with the presence of infection or inflammation in a wound, without physically removing the dressing. Our results show that the presence of NDs in the hybrid ND-silk membranes improves the thermal stability of silk fibers. NV- color centers in NDs embedded in silk fibers exhibit well-retained fluorescence and ODMR. Using the NV- centers as fluorescent nanoscale thermometers, we achieved temperature sensing in 25-50 °C, including the biologically relevant temperature window, for cell-grown ND-silk membranes. An enhancement (∼1.5× on average) in the temperature sensitivity of the NV- centers was observed for the hybrid materials. The hybrid membranes were further tested in vivo in a murine wound-healing model and demonstrated biocompatibility and equivalent wound closure rates as the control wounds. Additionally, the hybrid ND-silk membranes exhibited selective antifouling and biocidal propensity toward Gram-negative Pseudomonas aeruginosa and Escherichia coli, while no effect was observed on Gram-positive Staphylococcus aureus.

3D-Printed Diamond-Titanium Composite: A Hybrid Material for Implant Engineering.

Diamond-based implant materials make up an emerging research area where the materials could be prepared to promote cellular functions, decrease bacteria attachment, and be suitable for potential in situ imaging. Up until now, diamond implants have been fabricated using coating technologies or embedding diamond nanoparticles in polymer matrices. Here we demonstrated a method of manufacturing diamond implants using laser cladding technology to 3D print a composite of diamond and fused titanium material. Using this method, we could prepare composite scaffolds of up to 50% diamond, which has never been achieved before. We next investigated the interfacial properties of these scaffolds for potential applications in implants. The addition of diamond to the biomaterial results in a 30% decrease in the water contact angle, making the scaffolds more hydrophilic and improving cellular adhesion and proliferation.

Engineering the Interface: Nanodiamond Coating on 3D-Printed Titanium Promotes Mammalian Cell Growth and Inhibits Staphylococcus aureus Colonization.

Additively manufactured selective laser melted titanium (SLM-Ti) opens the possibility of tailored medical implants for patients. Despite orthopedic implant advancements, significant problems remain with regard to suboptimal osseointegration at the interface between the implant and the surrounding tissue. Here, we show that applying a nanodiamond (ND) coating onto SLM-Ti scaffolds provides an improved surface for mammalian cell growth while inhibiting colonization of Staphylococcus aureus bacteria. Owing to the simplicity of our methodology, the approach is suitable for coating SLM-Ti geometries. The ND coating achieved 32 and 29% increases in cell density of human dermal fibroblasts and osteoblasts, respectively, after 3 days of incubation compared with the uncoated SLM-Ti substratum. This increase in cell density complements an 88% reduction in S. aureus detected on the ND-coated SLM-Ti substrata. This study paves a way to create facile antifouling SLM-Ti structures for biomedical implants.

The effect of particle size on nanodiamond fluorescence and colloidal properties in biological media.

Fluorescent nanodiamonds (FNDs) are extremely photostable markers and nanoscale sensors, which are increasingly used in biomedical applications. Nanoparticle size is a critical parameter in the majority of these applications. Yet, the effect of particle size on FND's fluorescence and colloidal properties is not well understood today. Here, we investigate the fluorescence and colloidal stability of commercially available high-pressure high-temperature FNDs containing nitrogen-vacancy (NV) centers in biological media. Unconjugated FNDs in sizes ranging between 10 nm and 140 nm with an oxidized surface are studied using dynamic light scattering and fluorescence spectroscopy. We determine their colloidal stability in water, fetal bovine serum, Dulbecco's Modified Eagle Medium and complete media. The FNDs' relative fluorescence brightness, the NV charge-state, and the FND fluorescence against media autofluorescence are analyzed as a function of FND size. Our results will enable researchers in biology and beyond to identify the most promising FND particle size for their application.

UV plasmonic properties of colloidal liquid-metal eutectic gallium-indium alloy nanoparticles.

Nanoparticles made of non-noble metals such as gallium have recently attracted significant attention due to promising applications in UV plasmonics. To date, experiments have mostly focused on solid and liquid pure gallium particles immobilized on solid substrates. However, for many applications, colloidal liquid-metal nanoparticle solutions are vital. Here, we experimentally demonstrate strong UV plasmonic resonances of eutectic gallium-indium (EGaIn) liquid-metal alloy nanoparticles suspended in ethanol. We rationalise experimental results through a theoretical model based on Mie theory. Our results contribute to the understanding of UV plasmon resonances in colloidal liquid-metal EGaIn nanoparticle suspensions. They will also enable further research into emerging applications of UV plasmonics in biomedical imaging, sensing, stretchable electronics, photoacoustics, and electrochemistry.

High-fat diet worsens the impact of aging on microglial function and morphology in a region-specific manner.

Hippocampal microglia are vulnerable to the effects of aging, displaying a primed phenotype and hyper-responsiveness to various stimuli. We have previously shown that short-term high-fat diet (HFD) significantly impairs hippocampal- and amygdala-based cognitive function in the aged without affecting it in the young. Here, we assessed if morphological and functional changes in microglia might be responsible for this. We analyzed hippocampus and amygdala from young and aging rats that had been given three days HFD, a treatment sufficient to cause both hippocampal- and amygdala-dependent cognitive and neuroinflammatory differences in the aged. Aging led to the expected priming of hippocampal microglia in that it increased microglial numbers and reduced branching in this region. Aging also increased microglial phagocytosis of microbeads in the hippocampus, but the only effect of HFD in this region was to increase the presence of enlarged synaptophysin boutons in the aged, indicative of neurodegeneration. In the amygdala, HFD exacerbated the effects of aging on microglial priming (morphology) and markedly suppressed phagocytosis without notably affecting synaptophysin. These data reveal that, like the hippocampus, the amygdala displays aging-related microglial priming. However, the microglia in this region are also uniquely vulnerable to the detrimental effects of short-term HFD in aging.

Spiropyran-Based Nanocarrier: A New Zn2+ -Responsive Delivery System with Real-Time Intracellular Sensing Capabilities.

A new spiropyran-based stimuli-responsive delivery system is fabricated. It encapsulates and then releases an extraneous compound in response to elevated levels of Zn2+ , a critical factor in cell apoptosis. A C12 -alkyl substituent on the spiropyran promotes self-assembly into a micelle-like nanocarrier in aqueous media, with nanoprecipitation and encapsulation of added payload. Zn2+ binding occurs to an appended bis(2-pyridylmethyl)amine group at biologically relevant micromolar concentration. This leads to switching of the spiropyran (SP) isomer to the strongly fluorescent ring opened merocyanine-Zn2+ (MC-Zn2+ ) complex, with associated expansion of the nanocarriers to release the encapsulated payload. Payload release is demonstrated in solution and in HEK293 cells by encapsulation of a blue fluorophore, 7-hydroxycoumarin, and monitoring its release using fluorescence spectroscopy and microscopy. Furthermore, the use of the nanocarriers to deliver a caspase inhibitor, Azure B, into apoptotic cells in response to an elevated Zn2+ concentration is demonstrated. This then inhibits intracellular caspase activity, as evidenced by confocal microscopy and in real-time by time-lapsed microscopy. Finally, the nanocarriers are shown to release an encapsulated proteasome inhibitor (5) in Zn2+ -treated breast carcinoma cell line models. This then inhibits intracellular proteasome and induces cytotoxicity to the carcinoma cells.

Two-photon polymerisation 3D printed freeform micro-optics for optical coherence tomography fibre probes.

Miniaturised optical coherence tomography (OCT) fibre-optic probes have enabled high-resolution cross-sectional imaging deep within the body. However, existing OCT fibre-optic probe fabrication methods cannot generate miniaturised freeform optics, which limits our ability to fabricate probes with both complex optical function and dimensions comparable to the optical fibre diameter. Recently, major advances in two-photon direct laser writing have enabled 3D printing of arbitrary three-dimensional micro/nanostructures with a surface roughness acceptable for optical applications. Here, we demonstrate the feasibility of 3D printing of OCT probes. We evaluate the capability of this method based on a series of characterisation experiments. We report fabrication of a micro-optic containing an off-axis paraboloidal total internal reflecting surface, its integration as part of a common-path OCT probe, and demonstrate proof-of-principle imaging of biological samples.