X-ray Zernike phase contrast tomography: 3D ROI visualization of mm-sized mice organ tissues down to sub-cellular components.

Thanks to its non-invasive nature, X-ray phase contrast tomography is a very versatile imaging tool for biomedical studies. In contrast, histology is a well-established method, though having its limitations: it requires extensive sample preparation and it is quite time consuming. Therefore, the development of nano-imaging techniques for studying anatomic details at the cellular level is gaining more and more importance. In this article, full field transmission X-ray nanotomography is used in combination with Zernike phase contrast to image millimeter sized unstained tissue samples at high spatial resolution. The regions of interest (ROI) scans of different tissues were obtained from mouse kidney, spleen and mammalian carcinoma. Thanks to the relatively large field of view and effective pixel sizes down to 36 nm, this 3D approach enabled the visualization of the specific morphology of each tissue type without staining or complex sample preparation. As a proof of concept technique, we show that the high-quality images even permitted the 3D segmentation of multiple structures down to a sub-cellular level. Using stitching techniques, volumes larger than the field of view are accessible. This method can lead to a deeper understanding of the organs' nano-anatomy, filling the resolution gap between histology and transmission electron microscopy.

3D Imaging of Nanoparticle Distribution in Biological Tissue by Laser-Induced Breakdown Spectroscopy.

Nanomaterials represent a rapidly expanding area of research with huge potential for future medical applications. Nanotechnology indeed promises to revolutionize diagnostics, drug delivery, gene therapy, and many other areas of research. For any biological investigation involving nanomaterials, it is crucial to study the behavior of such nano-objects within tissues to evaluate both their efficacy and their toxicity. Here, we provide the first account of 3D label-free nanoparticle imaging at the entire-organ scale. The technology used is known as laser-induced breakdown spectroscopy (LIBS) and possesses several advantages such as speed of operation, ease of use and full compatibility with optical microscopy. We then used two different but complementary approaches to achieve 3D elemental imaging with LIBS: a volume reconstruction of a sliced organ and in-depth analysis. This proof-of-concept study demonstrates the quantitative imaging of both endogenous and exogenous elements within entire organs and paves the way for innumerable applications.

[Ultrasmall nanoparticles for radiotherapy: AGuIX]. / Nanoparticules ultrafines en radiothérapie : le cas des AGuIX.

Since twenty years, many nanoparticles based on high atomic number elements have been developed as radiosensitizers. The design of these nanoparticles is limited by the classical rules associated with the development of nanoparticles for oncology and by the specific ones associated to radiosensitizers, which aim to increase the effect of the dose in the tumor area and to spare the health tissues. For this application, systemic administration of nanodrugs is possible. This paper will discuss the development of AGuIX nanoparticles and will emphasize on this example the critical points for the development of a nanodrug for this application. AGuIX nanoparticles display hydrodynamic diameters of a few nanometers and are composed of polysiloxane and gadolinium chelates. This particle has been used in many preclinical studies and is evaluated for a further phase I clinical trial. Finally, in addition to its high radiosensitizing potential, AGuIX display MRI functionality and can be used as theranostic nanodrug for personalized medicine.

Laser spectrometry for multi-elemental imaging of biological tissues.

An increasing interest has arisen in research focused on metallic and organic ions that play crucial roles in both physiological and pathological metabolic processes. Current methods for the observation of trace elements in biological tissues at microscopic spatial resolution often require equipment with high complexity. We demonstrate a novel approach with an all-optical design and multi-elemental scanning imaging, which is unique among methods of elemental detection because of its full compatibility with standard optical microscopy. This approach is based on laser-induced breakdown spectroscopy (LIBS), which allows the elements in a tissue sample to be directly detected and quantified under atmospheric pressure. We successfully applied this method to murine kidneys with 10 µm resolution and a ppm-level detection limit to analyze the renal clearance of nanoparticles. These results offer new insight into the use of laser spectrometry in biomedical applications in the field of label-free elemental mapping of biological tissues.

The use of theranostic gadolinium-based nanoprobes to improve radiotherapy efficacy.

A new efficient type of gadolinium-based theranostic agent (AGuIX®) has recently been developed for MRI-guided radiotherapy (RT). These new particles consist of a polysiloxane network surrounded by a number of gadolinium chelates, usually 10. Owing to their small size (<5 nm), AGuIX typically exhibit biodistributions that are almost ideal for diagnostic and therapeutic purposes. For example, although a significant proportion of these particles accumulate in tumours, the remainder is rapidly eliminated by the renal route. In addition, in the absence of irradiation, the nanoparticles are well tolerated even at very high dose (10 times more than the dose used for mouse treatment). AGuIX particles have been proven to act as efficient radiosensitizers in a large variety of experimental in vitro scenarios, including different radioresistant cell lines, irradiation energies and radiation sources (sensitizing enhancement ratio ranging from 1.1 to 2.5). Pre-clinical studies have also demonstrated the impact of these particles on different heterotopic and orthotopic tumours, with both intratumoural or intravenous injection routes. A significant therapeutical effect has been observed in all contexts. Furthermore, MRI monitoring was proven to efficiently aid in determining a RT protocol and assessing tumour evolution following treatment. The usual theoretical models, based on energy attenuation and macroscopic dose enhancement, cannot account for all the results that have been obtained. Only theoretical models, which take into account the Auger electron cascades that occur between the different atoms constituting the particle and the related high radical concentrations in the vicinity of the particle, provide an explanation for the complex cell damage and death observed.

Conventional versus stealth lipid nanoparticles: formulation and in vivo fate prediction through FRET monitoring.

The determination of the nanocarrier fate in preclinical models is required before any translation from laboratory to clinical trials. Modern fluorescent imaging techniques have gained considerable advances becoming a powerful technology for non-invasive visualization in living subjects. Among them, Forster (fluorescence) resonance energy transfer (FRET) is a particular fluorescence imaging which involves energy transfer between 2 fluorophores in a distance-dependent manner. Considering this feature, the encapsulation of an acceptor/donor pair in lipid nanoparticles (LNEs: lipid nanoemulsions, LNCs: lipid nanocapsules) allowed the carrier integrity to be tracked. Accordingly, we used this FRET technique to evaluate the behavior of LNEs, conventional LNCs and newly designed stealth LNCs. After the development through a one-step (OS) PEGylation process of these stealth LNCs (OS LNCs), in vitro guest exchange dynamics and release kinetics were evaluated for both LNC formulations. We thereafter assessed in vivo biodistribution of all types of lipid nanoparticles. Results showed enhanced stability of encapsulation in OS LNCs in comparison to conventional LNCs. Additionally, the presence of the long PEG chains on the lipid nanoparticle surface altered the biodistribution pattern. Despite different release kinetic profiles, OS LNCs and LNEs showed extended blood circulation time associated with a good structure stability over several hours after intravenous injection.

Optical small animal imaging in the drug discovery process.

Molecular imaging of tumors in preclinical models is of the utmost importance for developing innovative cancer treatments. This field is moving extremely rapidly, with recent advances in optical imaging technologies and sophisticated molecular probes for in vivo imaging. The aim of this review is to provide a succinct overview of the imaging modalities available for rodents and with focus on describing optical probes for cancer imaging.

Drug development in oncology assisted by noninvasive optical imaging.

Early and accurate detection of tumors, like the development of targeted treatments, is a major field of research in oncology. The generation of specific vectors, capable of transporting a drug or a contrast agent to the primary tumor site as well as to the remote (micro-) metastasis would be an asset for early diagnosis and cancer therapy. Our goal was to develop new treatments based on the use of tumor-targeted delivery of large biomolecules (DNA, siRNA, peptides, or nanoparticles), able to induce apoptosis while dodging the specific mechanisms developed by tumor cells to resist this programmed cell death. Nonetheless, the insufficient effectiveness of the vectorization systems is still a crucial issue. In this context, we generated new targeting vectors for drug and biomolecules delivery and developed several optical imaging systems for the follow-up and evaluation of these vectorization systems in live mice. Based on our recent work, we present a brief overview of how noninvasive optical imaging in small animals can accelerate the development of targeted therapeutics in oncology.