We present a simple yet highly flexible 3D-model microsystem for the investigation of nanotherapeutics transport, ahead of in vivo studies, allowing to follow the penetration and distribution of nanoparticles within spheroids over space and time.
Abstract The huge gap between 2D in vitro assays used for drug screening, and the in vivo 3D-physiological environment hampered reliable predictions for the route and accumulation of nanotherapeutics in vivo. For such nanotherapeutics, Multi-Cellular Tumour Spheroids (MCTS) is emerging as a good alternative in vitro model. However, the classical approaches to produce MCTS suffer from low yield, slow process, difficulties in MCTS manipulation and compatibility with high-magnification fluorescent optical microscopy. On the other hand, spheroid-on-chip set-ups developed so far require a microfluidic practical knowledge difficult to transfer to a cell biology laboratory. We present here a simple yet highly flexible 3D-model microsystem consisting of agarose-based microwells. Fully compatible with the multi-well plates format conventionally used in cell biology, our simple process enables the formation of hundreds of reproducible spheroids in a single pipetting. Immunostaining and fluorescent imaging including live high-resolution optical microscopy can be performed in-situ , with no manipulation of spheroids. As a proof-of-principle of the relevance of such in vitro platform for nanotherapeutics evaluation, this study investigates the kinetic and localization of nanoparticles within colorectal cancer MCTS cells (HCT-116). The nanoparticles chosen are sub-5 nm ultrasmall nanoparticles made of polysiloxane and gadolinium chelates that can be visualized in MRI (AGuIX ® , currently implicated in clinical trials as effective radiosensitizers for radiotherapy) and confocal microscopy after addition of Cy 5.5. We show that the amount of AGuIX ® nanoparticles within cells is largely different in 2D and 3D. Using our flexible agarose-based microsystems, we are able to resolve spatially and temporally the penetration and distribution of AGuIX ® nanoparticles within MCTS. The nanoparticles are first found in both extracellular and intracellular space of MCTS. While the extracellular part is washed away after few days, we evidenced intracellular localisation of AGuIX ® , mainly within lysosomes compartment, but also occasionally within mitochondria. Our agarose-based microsystem appears hence as a promising 3D in vitro user-friendly platform for investigation of nanotherapeutics transport, ahead of in vivo studies. Abstract Figure Graphical abstract
Abstract In life sciences, there are increasing interest in 3D culture models to better reproduce the 3D environment encountered in-vivo. Imaging of such 3D culture models is instrumental for drug discovery, but face several issues before its use becomes widespread. Extensive microscopic investigation of these 3D cell models faces the challenge of light penetration in depth in opaque biological tissues. To overcome this limit, diverse clearing techniques have emerged over the past decades. However, it is not straightforward to choose the best clearing protocols, and assess quantitatively their clearing efficiency. Focusing on spheroids, we propose a combination of fast and cost-effective clearing procedure for such medium-sized samples. A generic method with local contrast metrics and deep convolutional neural network-based segmentation of nuclei is proposed to quantify the efficiency of clearing. We challenged this method by testing the possibility to transfer segmentation knowledge from a clearing protocol to another. The later results support the pertinence of training deep learning algorithms on cleared samples to further use the segmentation pipeline on non-cleared ones. This second step of the protocol gives access to digital clearing possibilities applicable to live and high-throughput optical imaging.
Correction for 'Quantifying nanotherapeutic penetration using a hydrogel-based microsystem as a new 3D in vitro platform' by Saba Goodarzi et al., Lab Chip, 2021, 21, 2495-2510, DOI: 10.1039/D1LC00192B.
Titanium alloys have been extensively used as promising implant materials. The anodic oxide layer on the surface of this alloy can be a compact, porous or a tubular structure, which has a direct impact on the final characteristics of the implants. In this study, nano topographic oxide arrays were synthesized on the surface of titanium substrates using an anodic oxidation technique. The anodization process was performed at a two-electrode electrochemical cell, and then the samples were annealed to obtain crystalline structures. The synthesized samples were analyzed to evaluate the compositional phase, morphology, surface hydrophilicity and corrosion resistance of the nanostructured oxide arrays in artificial saliva. Microscopic observations confirmed the formation of a nanotubular structure on the surface of titanium substrate depending on the anodization condition. After heat-treatment at 570 °C, crystallographic analyses showed that the obtained crystalline phase was a mixture of Anatase and Rutile phases. The electrochemical impedance spectroscopy (EIS) results indicated a significant improvement in the corrosion resistance and electrochemical stability of the anodized sample in artificial saliva compare to the control samples. In addition, the anodized samples showed a better hydrophilic characteristics, viability and proliferation of periodontal ligament cells in comparison with the un-anodized samples. This study demonstrated that the anodized titanium samples, with the nanotubular structure on the surfaces, could be considered as a good candidate for dental implants.
In 1985, the serendipitous discovery of fullerene triggered the research of carbon structures into the world of symmetric nanomaterials. Consequently, Robert F. Curl, Harold W. Kroto and Richard E. Smalley were awarded the Noble prize in chemistry for their discovery of the buckminsterfullerene (C60 with a cage-like fused-ring structure). Fullerene, as the first symmetric nanostructure in carbon nanomaterials family, opened up new perspectives in nanomaterials field leading to discovery and research on other symmetric carbon nanomaterials like carbon nanotubes and two-dimensional graphene which put fullerenes in the shade, while fullerene as the most symmetrical molecule in the world with incredible properties deserves more attention in nanomaterials studies. Buckyball with its unique structure consisting of sp2 carbons which form a high symmetric cage with different sizes (C60, C70 and so on); however, the most abundant among them is C60 which possesses 60 carbon atoms. The combination of unique properties of this molecule extends its applications in divergent areas of science, especially those related to biomedical engineering. This review aims to be a comprehensive review with a broad interest to the biomedical engineering community, being a substantial overview of the most recent advances on fullerenes in biomedical applications that have not been exhaustively and critically reviewed in the past few years.
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