Correlative Fluorescence and Transmission Electron Microscopy Assisted by 3D Machine Learning Reveals Thin Nanodiamonds Fluoresce Brighter.

Nitrogen vacancy (NV) centers in fluorescent nanodiamonds (FNDs) draw widespread attention as quantum sensors due to their room-temperature luminescence, exceptional photo- and chemical stability, and biocompatibility. For bioscience applications, NV centers in FNDs offer high-spatial-resolution capabilities that are unparalleled by other solid-state nanoparticle emitters. On the other hand, pursuits to further improve the optical properties of FNDs have reached a bottleneck, with intense debate in the literature over which of the many factors are most pertinent. Here, we describe how substantial progress can be achieved using a correlative transmission electron microscopy and photoluminescence (TEMPL) method that we have developed. TEMPL enables a precise correlative analysis of the fluorescence brightness, size, and shape of individual FND particles. Augmented with machine learning, TEMPL can be used to analyze a large, statistically meaningful number of particles. Our results reveal that FND fluorescence is strongly dependent on particle shape, specifically, that thin, flake-shaped particles are up to several times brighter and that fluorescence increases with decreasing particle sphericity. Our theoretical analysis shows that these observations are attributable to the constructive interference of light waves within the FNDs. Our findings have significant implications for state-of-the-art sensing applications, and they offer potential avenues for improving the sensitivity and resolution of quantum sensing devices.

Complex Dispersion of Detonation Nanodiamond Revealed by Machine Learning Assisted Cryo-TEM and Coarse-Grained Molecular Dynamics Simulations.

Understanding the polydispersity of nanoparticles is crucial for establishing the efficacy and safety of their role as drug delivery carriers in biomedical applications. Detonation nanodiamonds (DNDs), 3-5 nm diamond nanoparticles synthesized through detonation process, have attracted great interest for drug delivery due to their colloidal stability in water and their biocompatibility. More recent studies have challenged the consensus that DNDs are monodispersed after their fabrication, with their aggregate formation poorly understood. Here, we present a novel characterization method of combining machine learning with direct cryo-transmission electron microscopy imaging to characterize the unique colloidal behavior of DNDs. Together with small-angle X-ray scattering and mesoscale simulations we show and explain the clear differences in the aggregation behavior between positively and negatively charged DNDs. Our new method can be applied to other complex particle systems, which builds essential knowledge for the safe implementation of nanoparticles in drug delivery.

On the growth of the soft and hard protein corona of mesoporous silica particles with varying morphology.

The characterization of the protein corona has become an essential part of understanding the biological properties of nanomaterials. This is also important in the case of mesoporous silica particles intended for use as drug delivery excipients. A combination of scattering, imaging and protein characterization techniques is used here to assess the effect of particle shape and growth of the reversible (soft) and strongly bound (hard) corona of three types mesoporous silica particles with different aspect ratios. Notable differences in the protein composition, surface coverage and particle agglomeration of the protein corona-particle complex point to specific protein adsorption profiles highly dependent on exposed facets and aspect ratio. Spherical particles form relatively homogeneous soft and hard protein coronas (approx.10 nm thick) with higher albumin content. In contrast to rod-shaped and faceted particles, which possess soft coronas weakly bound to the external surface and influenced to a greater extent by the particle morphology. These differences are likely important contributors to observed changes in biological properties, such as cell viability and immunological behaviour, with mesoporous silica particle shape.

Steric Interaction of Polyglycerol-Functionalized Detonation Nanodiamonds.

Detonation nanodiamonds have found numerous potential applications in a diverse array of fields such as biomedical imaging and drug delivery. Here, we systematically characterized non-functionalized and polyglycerol-functionalized detonation nanodiamond particles (DNPs) dispersed in aqueous suspensions at different ionic strengths (∼1.0 × 10-7 to 1.0 × 10-2 M) via dynamic light scattering and cryogenic transmission electron microscopy. For these colloidal suspensions, the total potential energies of interactions between a pair of DNPs were theoretically calculated using the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory plus the fitting of the Boltzmann distribution to the interparticle spacing distribution of the colloidal DNPs. These investigations revealed that the non-functionalized DNPs are dispersed in aqueous media through the long-range (>10 nm) and weak (<7 kBT) electrical double-layer repulsive interaction, while the driving force on dispersion of polyglycerol-functionalized DNPs is mostly derived from the short-range (<2 nm) and strong (∼55 kBT) steric repulsive potential barrier generated by the polyglycerol. Moreover, our results show that the truly monodispersed and individually dispersed DNP colloids, forming no aggregates in aqueous suspensions, are available by both functionalizing DNPs by polyglycerol and increasing ionic strength of suspending media to ≳1.0 × 10-2 M.

Chick Embryo Experimental Platform for Micrometastases Research in a 3D Tissue Engineering Model: Cancer Biology, Drug Development, and Nanotechnology Applications.

Colonization of distant organs by tumor cells is a critical step of cancer progression. The initial avascular stage of this process (micrometastasis) remains almost inaccessible to study due to the lack of relevant experimental approaches. Herein, we introduce an in vitro/in vivo model of organ-specific micrometastases of triple-negative breast cancer (TNBC) that is fully implemented in a cost-efficient chick embryo (CE) experimental platform. The model was built as three-dimensional (3D) tissue engineering constructs (TECs) combining human MDA-MB-231 cells and decellularized CE organ-specific scaffolds. TNBC cells colonized CE organ-specific scaffolds in 2-3 weeks, forming tissue-like structures. The feasibility of this methodology for basic cancer research, drug development, and nanomedicine was demonstrated on a model of hepatic micrometastasis of TNBC. We revealed that MDA-MB-231 differentially colonize parenchymal and stromal compartments of the liver-specific extracellular matrix (LS-ECM) and become more resistant to the treatment with molecular doxorubicin (Dox) and Dox-loaded mesoporous silica nanoparticles than in monolayer cultures. When grafted on CE chorioallantoic membrane, LS-ECM-based TECs induced angiogenic switch. These findings may have important implications for the diagnosis and treatment of TNBC. The methodology established here is scalable and adaptable for pharmacological testing and cancer biology research of various metastatic and primary tumors.

Gold Nanostars with Reduced Fouling Facilitate Small Molecule Detection in the Presence of Protein.

Gold nanoparticles have the potential to be used in biomedical applications from diagnostics to drug delivery. However, interactions of gold nanoparticles with different biomolecules in the cellular environment result in the formation of a "protein corona"-a layer of protein formed around a nanoparticle, which induces changes in the properties of nanoparticles. In this work we developed methods to reproducibly synthesize spheroidal and star-shaped gold nanoparticles, and carried out a physico-chemical characterization of synthesized anionic gold nanospheroids and gold nanostars through transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential (ZP), nanoparticles tracking analysis (NTA), ultraviolet-visible (UV-Vis) spectroscopy and estimates of surface-enhanced Raman spectroscopy (SERS) signal enhancement ability. We analyzed how they interact with proteins after pre-incubation with bovine serum albumin (BSA) via UV-Vis, DLS, ZP, NTA, SERS, cryogenic TEM (cryo-TEM) and circular dichroism (CD) spectroscopy. The tests demonstrated that the protein adsorption on the particles' surfaces was different for spheroidal and star shaped particles. In our experiments, star shaped particles limited the protein corona formation at SERS "hot spots". This benefits the small-molecule sensing of nanostars in biological media. This work adds more understanding about protein corona formation on gold nanoparticles of different shapes in biological media, and therefore guides design of particles for studies in vitro and in vivo.

A lysozyme corona complex for the controlled pharmacokinetic release of probucol from mesoporous silica particles.

Mesoporous silica particles (MSPs) enhance the release kinetics of poorly soluble compound probucol (PB) under the influence of a pore-blocking protein corona, prepared with lysozyme protein adsorption. In vivo oral administration experiments show a prolongation in the time to reach maximum systemic concentration and half-life of PB released from the lysozyme-MSP complex in comparison to the MSP alone. Specific hard protein corona complexes can act as functional diffusion barriers for the controlled release of drugs from MSP based formulations.

Effect of a protein corona on the fibrinogen induced cellular oxidative stress of gold nanoparticles.

The protein corona of nanoparticles is becoming a tool to understand the relation between intrinsic physicochemical properties and extrinsic biological behaviour. A diverse set of characterisation techniques such as transmission electron microscopy, mass spectrometry, dynamic light scattering, zeta-potential measurements and surface enhanced Raman spectroscopy are used to determine the composition and physical properties of the soft and hard corona formed around spherical gold nanoparticles. Advanced characterisation via small angle X-ray scattering and cryo-transmission electron microscopy suggests the presence of a thin hard corona of a few nm on 50 nm gold nanoparticles. The protein corona does not cause changes in cell viability, but inhibits the generation of reactive oxygen species in microglia cells. When a pre-incubated layer of fibrinogen, a protein with high affinity for the gold surface, is present around the nanoparticles before a protein corona is formed in bovine serum, the cellular uptake is significantly increased with an inhibition of ROS. The selective sequential pre-formation of protein complexes prior to incubation in cells is demonstrated as a viable method to alter the biological behaviour of nanoparticles.