In Vivo Ultrasound Imaging of Macrophages Using Acoustic Vaporization of Internalized Superheated Nanodroplets.

Activating patients' immune cells, either by reengineering them or treating them with bioactive molecules, has been a breakthrough in the field of immunotherapy and has revolutionized treatment, especially against cancer. As immune cells naturally home to tumors or injured tissues, labeling such cells holds promise for non-invasive tracking and biologic manipulation. Our study demonstrates that macrophages loaded with extremely low boiling point perfluorocarbon nanodroplets not only survive ultrasound-induced phase change but also maintain their phagocytic function. Unlike observations made when using higher boiling point perfluorocarbon nanodroplets, our results show that phase change occurs intracellularly at a low mechanical index using a clinical scanner operating within the energy limit set by the Food and Drug Administration (FDA). After nanodroplet-loaded macrophages were given intravenously to nude rats, they were invisible in the liver when imaged at a very low mechanical index using a clinical ultrasound scanner. They became visible when power was increased but still within the FDA limits up to 8 h after administration. The acoustic labeling and in vivo detection of macrophages using a clinical ultrasound scanner represent a paradigm shift in the field of cell tracking and pave the way for potential therapeutic strategies in the clinical setting.

Cancer immunotherapy based on image-guided STING activation by nucleotide nanocomplex-decorated ultrasound microbubbles.

The cytosolic innate immune sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is crucial for priming adaptive antitumour immunity through antigen-presenting cells (APCs). Natural agonists, such as cyclic dinucleotides (CDNs), activate the cGAS-STING pathway, but their clinical translation is impeded by poor cytosolic entry and serum stability, low specificity and rapid tissue clearance. Here we developed an ultrasound (US)-guided cancer immunotherapy platform using nanocomplexes composed of 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) electrostatically bound to biocompatible branched cationic biopolymers that are conjugated onto APC-targeting microbubbles (MBs). The nanocomplex-conjugated MBs engaged with APCs and efficiently delivered cGAMP into the cytosol via sonoporation, resulting in activation of cGAS-STING and downstream proinflammatory pathways that efficiently prime antigen-specific T cells. This bridging of innate and adaptive immunity inhibited tumour growth in both localized and metastatic murine cancer models. Our findings demonstrate that targeted local activation of STING in APCs under spatiotemporal US stimulation results in systemic antitumour immunity and improves the therapeutic efficacy of checkpoint blockade, thus paving the way towards novel image-guided strategies for targeted immunotherapy of cancer.

Catalase-Loaded Silica Nanoparticles Formulated via Direct Surface Modification as Potential Oxygen Generators for Hypoxia Relief.

Enzymes are biological catalysts that have many potential industrial and biomedical applications. However, the widespread use of enzymes in the industry has been limited by their instability and poor recovery. In biomedical applications, systemic administration of enzymes has faced two main challenges: limited bioactivity mostly due to rapid degradation by proteases and immunogenic activity, since most enzymes are from nonhuman sources. Herein, we propose a robust enzyme-encapsulation strategy to mitigate these limitations. Catalase (CAT) was encapsulated in nanoporous silica nanoparticles (CAT-SiNPs) by first chemically modifying the enzyme surface with a silica precursor, followed by silica growth and finally poly(ethylene glycol) (PEG) conjugation. The formulation was carried out in mild aqueous conditions and yielded nanoparticles (NPs) with a mean diameter of 230 ± 10 nm and a concentration of 1.3 ± 0.8 × 1012 NPs/mL. CAT-SiNPs demonstrated high enzyme activity, optimal protection from proteolysis by proteinase K and trypsin, and excellent stability over time. In addition, a new electrochemical assay was developed to measure CAT activity in a rapid, simple, and accurate manner without interference from chromophore usually present in biological samples. Concentrations of 2.5 × 1010 to 80 × 1010 CAT-SiNPs/mL not only proved to be nontoxic in cell cultures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay but also conferred cell protection when cells were exposed to 1 mM hydrogen peroxide (H2O2). Finally, the ability of CAT-SiNPs to release oxygen (O2) when exposed to H2O2 was demonstrated in vivo using a rat model. Following the direct injection of CAT-SiNPs in the left kidney, partial pressure of oxygen (pO2) increased by more than 30 mmHg compared to the contralateral control kidney during the systemic infusion of safe levels of H2O2. This pilot study highlights the potential of CAT-SiNPs to generate O2 to relieve hypoxia in tissues and potentially sensitize tumors against radiation therapy.

Multimodal gadolinium oxysulfide nanoparticles for bioimaging: A comprehensive biodistribution, elimination and toxicological study.

For some years now, gadolinium oxysulfide nanoparticles (NPs) appear as strong candidates for very efficient multimodal in vivo imaging by: 1) Magnetic Resonance (MRI), 2) X-ray Computed Tomography (CT) and 3) photoluminescence imaging. In this paper, we present a selection of results centered on the evaluation of physico-chemical stability, toxicity, bio-distribution and excretion mechanisms of Gd2O2S:Ln3+ nanoparticles intravenously injected in rats. Two formulations are here tested with a common matrix and different dopants: Gd2O2S:Eu3+5% and Gd2O2S:Yb3+4%/Tm3+0.1%. The NPs appear to be almost insoluble in pure water and human plasma but corrosion/degradation phenomenon appears in acidic conditions classically encountered in cell lysosomes. Whole body in vivo distribution, excretion and toxicity evaluation revealed a high tolerance of nanoparticles with a long-lasting imaging signal associated with a slow hepatobiliary clearance and very weak urinary excretion. The results show that the majority of the injected product (>60%) has been excreted through the feces after five months. Experiments have evidenced that the NPs mainly accumulate in macrophage-rich organs, that is mainly liver and spleen and to a lesser extent lungs and bones (mainly marrow). No significant amounts have been detected in other organs such as heart, kidneys, brain, intestine and skin. Gd2O2S:Ln3+ NPs appeared to be very well tolerated up to 400 mg/kg when administered intravenously. STATEMENT OF SIGNIFICANCE: Since 2011, we have focused our work on Gd2O2S nanoparticles (NPs) for multimodal bioimaging using fluorescence, Magnetic Resonance Imaging (MRI) and Computed Tomography with very efficient results already published. However, since the European Medicines Agency has concluded its review of gadolinium contrast agents, confirming recommendations to restrict the use of some linear gadolinium agents used in MRI, a particular attention must be paid to any new contrast media containing gadolinium. Therefore, we present in this paper a compilation of studies about toxicity, bio-distribution and excretion mechanisms of Gd2O2S:Ln3+ NPs intravenously injected into rats. We also present an in vitro kinetic study of NPs degradation in aqueous and biological media to provide some information on chemical and biological stability.

Evaluation of upconverting nanoparticles towards heart theranostics.

Restricted and controlled drug delivery to the heart remains a challenge giving frequent off-target effects as well as limited retention of drugs in the heart. There is a need to develop and optimize tools to allow for improved design of drug candidates for treatment of heart diseases. Over the last decade, novel drug platforms and nanomaterials were designed to confine bioactive materials to the heart. Yet, the research remains in its infancy, not only in the development of tools but also in the understanding of effects of these materials on cardiac function and tissue integrity. Upconverting nanoparticles are nanomaterials that recently accelerated interest in theranostic nanomedicine technologies. Their unique photophysical properties allow for sensitive in vivo imaging that can be combined with spatio-temporal control for targeted release of encapsulated drugs. Here we synthesized upconverting NaYF4:Yb,Tm nanoparticles and show for the first time their innocuity in the heart, when injected in the myocardium or in the pericardial space in mice. Nanoparticle retention and upconversion in the cardiac region did not alter heart rate variability, nor cardiac function as determined over a 15-day time course ensuing the sole injection. Altogether, our nanoparticles show innocuity primarily in the pericardial region and can be safely used for controlled spatiotemporal drug delivery. Our results support the use of upconverting nanoparticles as potential theranostics tools overcoming some of the key limitations associated with conventional experimental cardiology.

Multimodal gadolinium oxysulfide nanoparticles: a versatile contrast agent for mesenchymal stem cell labeling.

Despite a clear development of innovative therapies based on stem cell manipulation, the availability of new tools to better understand and follow stem cell behavior and improve their biomedical applications is not adequate. Indeed, an ideal tracking device must have good ability to label stem cells as well as complete neutrality relative to their biology. Furthermore, preclinical studies imply in vitro and in vivo approaches that often require several kinds of labeling and/or detection procedures. Consequently, the multimodality concept presented in this work may present a solution to this problem as it has the potential to combine complementary imaging techniques. Spherical europium-doped gadolinium oxysulfide (Gd2O2S:Eu3+) nanoparticles are presented as a candidate as they are detectable by (1) magnetic resonance (MRI), (2) X-ray and (3) photoluminescence imaging. Whole body in vivo distribution, elimination and toxicity evaluation revealed a high tolerance of nanoparticles with a long-lasting MRI signal and slow hepatobiliary and renal clearance. In vitro labeling of a wide variety of cells unveils the nanoparticle potential for efficient and universal cell tracking. Emphasis on mesenchymal stromal cells (MSCs) leads to the definition of optimal conditions for labeling and tracking in the context of cell therapy: concentrations below 50 µg mL-1 and diameters between 170 and 300 nm. Viability, proliferation, migration and differentiation towards mesodermal lineages are preserved under these conditions, and cell labeling appears to be persistent and without any leakage. Ex vivo detection of as few as five thousand Gd2O2S:Eu3+-labeled MSCs by MRI combined with in vitro examination with fluorescence microscopy highlights the feasibility of cell tracking in cell therapy using this new nanoplatform.