Achraf Al Faraj

In vivo biodistribution and biological impact of injected carbon nanotubes using magnetic resonance techniques

Background: Single-walled carbon nanotubes (SWCNT) hold promise for applications as contrast agents and target delivery carriers in the field of nanomedicine. When administered in vivo, their biodistribution and pharmacological profile needs to be fully characterized. The tissue distribution of carbon nanotubes and their potential impact on metabolism depend on their shape, coating, and metallic impurities. Because standard radiolabeled or fluorescently-labeled pharmaceuticals are not well suited for long-term in vivo follow-up of carbon nanotubes, alternative methods are required. Methods: In this study, noninvasive in vivo magnetic resonance imaging (MRI) investigations combined with high-resolution magic angle spinning (HR-MAS), Raman spectroscopy, iron assays, and histological analysis ex vivo were proposed and applied to assess the biodistribution and biological impact of intravenously injected pristine (raw and purified) and functionalized SWCNT in a 2-week longitudinal study. Iron impurities allowed raw detection of SWCNT in vivo by susceptibility-weighted MRI. Results: A transitional accumulation in the spleen and liver was observed by MRI. Raman spectroscopy, iron assays, and histological findings confirmed the MRI readouts. Moreover, no acute toxicological effect on the liver metabolic profile was observed using the HR-MAS technique, as confirmed by quantitative real-time polymerase chain reaction analysis. Conclusion: This study illustrates the potential of noninvasive MRI protocols for longitudinal assessment of the biodistribution of SWCNT with associated intrinsic metal impurities. The same approach can be used for any other magnetically-labeled nanoparticles.

Magnetic single-walled carbon nanotubes as efficient drug delivery nanocarriers in breast cancer murine model: noninvasive monitoring using diffusion-weighted magnetic resonance imaging as sensitive imaging biomarker.

Purpose Targeting doxorubicin (DOX) by means of single-walled carbon nanotube (SWCNT) nanocarriers may help improve the clinical utility of this highly active therapeutic agent. Active targeting of SWCNTs using tumor-specific antibody and magnetic attraction by tagging the nanotubes with iron oxide nanoparticles can potentially reduce the unnecessary side effects and provide enhanced theranostics. In the current study, the in vitro and in vivo efficacy of DOX-loaded SWCNTs as theranostic nanoprobes was evaluated in a murine breast cancer model. Methods Iron-tagged SWCNTs conjugated with Endoglin/CD105 antibody with or without DOX were synthetized and extensively characterized. Their biocompatibility was assessed in vitro in luciferase (Luc2)-expressing 4T1 (4T1-Luc2) murine breast cancer cells using TiterTACS™ Colorimetric Apoptosis Detection Kit (apoptosis induction), poly (ADP-ribose) polymerase (marker for DNA damage), and thiobarbituric acid-reactive substances (oxidative stress generation) assays, and the efficacy of DOX-loaded SWCNTs was evaluated by measuring the radiance efficiency using bioluminescence imaging (BLI). Tumor progression and growth were monitored after 4T1-Luc2 cells inoculation using noninvasive BLI and magnetic resonance imaging (MRI) before and after subsequent injection of SWCNT complexes actively and magnetically targeted to tumor sites. Results Significant increases in apoptosis, DNA damage, and oxidative stress were induced by DOX-loaded SWCNTs. In addition, a tremendous decrease in bioluminescence was observed in a dose- and time-dependent manner. Noninvasive BLI and MRI revealed successful tumor growth and subsequent attenuation along with metastasis inhibition following DOX-loaded SWCNTs injection. Magnetic tagging of SWCNTs was found to produce significant discrepancies in apparent diffusion coefficient values providing a higher contrast to detect treatment-induced variations as noninvasive imaging biomarker. In addition, it allowed their sensitive noninvasive diagnosis using susceptibility-weighted MRI and their magnetic targeting using an externally applied magnet. Conclusion Enhanced therapeutic efficacy of DOX delivered through antibody-conjugated magnetic SWCNTs was achieved. Further, the superiority of apparent diffusion coefficient measurements using diffusion-weighted MRI was found to be a sensitive imaging biomarker for assessment of treatment-induced changes.

Endothelial Cell–derived Microparticles Loaded with Iron Oxide Nanoparticles: Feasibility of MR Imaging Monitoring in Mice

PURPOSEnTo assess the feasibility of loading iron oxide nanoparticles in endothelial microparticles (EMPs), thereby enabling their noninvasive monitoring with magnetic resonance (MR) imaging in mice.nnnMATERIALS AND METHODSnExperiments were approved by the French Ministry of Agriculture. Endothelial cells, first labeled with anionic superparamagnetic nanoparticles, were stimulated to generate EMPs, carrying the nanoparticles in their inner compartment. C57BL/6 mice received an intravenous injection of nanoparticle-loaded EMPs, free nanoparticles, or the supernatant of nanoparticle-loaded EMPs. A 1-week follow-up was performed with a 4.7-T MR imaging device by using a gradient-echo sequence for imaging spleen, liver, and kidney and a radial very-short-echo time sequence for lung imaging. Comparisons were performed by using the Student t test.nnnRESULTSnThe signal intensity loss induced by nanoparticle-loaded EMPs or free nanoparticles was readily detected within 5 minutes after injection in the liver and spleen, with a more pronounced effect in the spleen for the magnetic EMPs. The kinetics of signal intensity attenuation differed for nanoparticle-loaded EMPs and free nanoparticles. No signal intensity changes were observed in mice injected with the supernatant of nanoparticle-loaded EMPs, confirming that cells had not released free nanoparticles, but only in association with EMPs. The results were confirmed by using Perls staining and immunofluorescence analysis.nnnCONCLUSIONnThe strategy to generate EMPs with magnetic properties allowed noninvasive MR imaging assessment and follow-up of EMPs and opens perspectives for imaging the implications of these cellular vectors in diseases.

Combination of drug-conjugated SWCNT nanocarriers for efficient therapy of cancer stem cells in a breast cancer animal model

Targeting breast cancer and more specifically cancer stem cell (CSC) subpopulation, responsible for tumor growth, resistance and self-renewal, using combination of therapeutic drugs selectively delivered via biocompatible nanocarriers, provides a novel approach for effective therapy. Here, we propose to evaluate the potential therapeutic efficacy of combining Paclitaxel and Salinomycin drugs actively targeted to both breast cancer and CSCs in xenograft murine model after conjugation with biocompatible CD44 antibody conjugated SWCNTs via hydrazone linker allowing pH-responsive release mechanism near the acidic tumor microenvironment. Both in vitro investigations on MDA-MB-231, sorted CSC negative or CSC positive fractions and in vivo evaluations on tumor-bearing mice using noninvasive bioluminescence and magnetic resonance imaging confirmed the enhanced therapeutic effect of the combined therapy compared to treatment with individual drug-conjugated nanocarriers or free drug suspensions. Thus, confirmed the great promise of the developed SWCNTs drug delivery system for effective breast cancer treatment by targeting and eradicating both whole tumor cells and CSCs populations.

Real-time high-resolution magnetic resonance tracking of macrophage subpopulations in a murine inflammation model: a pilot study with a commercially available cryogenic probe.

Macrophages present different polarization states exhibiting distinct functions in response to environmental stimuli. However, the dynamic of their migration to sites of inflammation is not fully elucidated. Here we propose a real-time in vivo cell tracking approach, using high-resolution (HR)-MRI obtained with a commercially available cryogenic probe (Cryoprobe™), to monitor trafficking of differently polarized macrophages after systemic injection into mice. Murine bone marrow-derived mononuclear cells were differentiated ex vivo into nonpolarized M0, pro-inflammatory M1 and immunomodulator M2 macrophage subsets and labeled with citrate-coated anionic iron oxide nanoparticles (AMNP). These cells were subsequently intravenously injected to mice bearing calf muscle inflammation. Whole body migration dynamics of macrophage subsets was monitored by MRI at 4.7u2009T with a volume transmission/reception radiofrequency coil and macrophage infiltration to the inflamed paw was monitored with the cryogenic probe, allowing 3D spatial resolution of 50u2009µm with a scan time of only 10u2009min. Capture of AMNP was rapid and efficient regardless of macrophage polarization, with the highest uptake in M2 macrophages. Flow cytometry confirmed that macrophages preserved their polarization hallmarks after labeling. Migration kinetics of labeled cells differed from that of free AMNP. A preferential homing of M2-polarized macrophages to inflammation sites was observed. Our in vivo HR-MRI protocol highlights the extent of macrophage infiltration to the inflammation site. Coupled to whole body imaging, HR-MRI provides quantitative information on the time course of migration of ex vivo-polarized intravenously injected macrophages.

Preferential Macrophage Recruitment and Polarization in LPS-Induced Animal Model for COPD: Noninvasive Tracking Using MRI

Noninvasive imaging of macrophages activity has raised increasing interest for diagnosis of chronic obstructive respiratory diseases (COPD), which make them attractive vehicles to deliver contrast agents for diagnostic or drugs for therapeutic purposes. This study was designed to monitor and evaluate the migration of differently polarized M1 and M2 iron labeled macrophage subsets to the lung of a LPS-induced COPD animal model and to assess their polarization state once they have reached the inflammatory sites in the lung after intravenous injection. Ex vivo polarized bone marrow derived M1 or M2 macrophages were first efficiently and safely labeled with amine-modified PEGylated dextran-coated SPIO nanoparticles and without altering their polarization profile. Their biodistribution in abdominal organs and their homing to the site of inflammation in the lung was tracked for the first time using a free-breathing non-invasive MR imaging protocol on a 4.7T magnet after their intravenous administration. This imaging protocol was optimized to allow both detection of iron labeled macrophages and visualization of inflammation in the lung. M1 and M2 macrophages were successfully detected in the lung starting from 2 hours post injection with no variation in their migration profile. Quantification of cytokines release, analysis of surface membrane expression using flow cytometry and immunohistochemistry investigations confirmed the successful recruitment of injected iron labeled macrophages in the lung of COPD mice and revealed that even with a continuum switch in the polarization profile of M1 and M2 macrophages during the time course of inflammation a balanced number of macrophage subsets predominate.

Specific targeting and noninvasive imaging of breast cancer stem cells using single-walled carbon nanotubes as novel multimodality nanoprobes

BACKGROUNDnThe limitation of current breast cancer treatments was elucidated by the presence of cancer stem cells (CSCs) that play essential role in cancer initiation, progression, resistance, recurrence and metastasis. Materialsxa0& methods: Biocompatible multimodality single-walled carbon nanotube (SWCNT) nanoprobes were developed. The biodistribution and preferential homing of CD44 antibody-conjugated SWCNTs toward the tumor site were monitored using MRI, single-photon emission computed tomography and near-infrared fluorescence imaging noninvasive imaging modalities.nnnRESULTSnQuantification of SWCNTs by sensitively measuring iron content in sorted CSC populations using inductively coupled plasma-mass spectrometry confirmed the enhanced selective targeting of anti-CD44 SWCNT and immunohistochemistry analyses revealed enhanced colocalization with areas rich in CD44 receptors.nnnDISCUSSION & CONCLUSIONnThis preclinical study provided encouraging results for efficient targeting of breast CSCs and perspectives for further clinical studies to confirm the efficacy and safety of the designed nanocarriers.

Preferential magnetic targeting of carbon nanotubes to cancer sites: noninvasive tracking using MRI in a murine breast cancer model

AIMnThis study evaluated the improvement in magnetic targeting of single-walled carbon nanotubes (SWCNTs) in a 4T1-induced breast cancer murine model and compared their enhanced delivery with active targeted SWCNTs conjugated with a specific antibody for prospective applications as drug-delivery nanocarriers.nnnMATERIALS & METHODSnPolyvinylpyrrolidone SWCNTs, loaded with iron oxide nanoparticles to improve their magnetic resonance detection and magnet attraction using an optimized flexible magnet positioned over the tumor site were developed. They were equally conjugated with Endoglin/CD105 antibody for SWCNTs active targeting. A noninvasive MRI protocol was then optimized to allow in vivo imaging of tumor site, sensitive detection of SWCNTs and apparent diffusion coefficient measurements. Special focus was devoted to evaluate the biocompatibility of the used SWCNTs.nnnRESULTSnIron-tagged SWCNTs exhibited very high magnetic resonance r2* relaxivities allowing their sensitive detection using noninvasive MRI and enhanced targeting using the magnet. Biocompatibility evaluations confirmed their safety for animal administration. Both T2* and apparent diffusion coefficient measurements confirmed their enhanced magnetic targeting starting from 2 h postinjection while a lower, but statistically significant enhanced targeting of antibody-conjugated active targeting was observed starting from 24 h postinjection of iron-tagged SWCNT + CD105 samples.nnnCONCLUSIONnThese results demonstrate the efficiency of magnetic targeting to specifically deliver higher load of iron-tagged SWCNTs as novel nanocarriers for cancer theranostics and allow their sensitive detection using noninvasive MRI.

Preferential magnetic nanoparticle uptake by bone marrow derived macrophages sub-populations: effect of surface coating on polarization, toxicity, and in vivo MRI detection

Noninvasive imaging of macrophages activity has raised increasing interest for diagnosis of different diseases, which make them attractive vehicles to deliver contrast agents or drugs for diagnostic or therapeutic purposes. In this study, the effect of polyethylene glycol functionalization of magnetic iron oxide nanoparticles and their further surface modification with carboxylic groups on bone marrow-derived M1 and M2 macrophages phenotype, labeling efficiency, uptake mechanism, biocompatibility, and their in vivo MR detection was assessed. An enhanced labeling efficiency was observed for carboxylic surface-modified superparamagnetic iron oxide (SPIO) compared to PEGylated SPIO and to a higher extent to plain SPIO along with a higher uptake by M2 subsets. Magnetic nanoparticles were found located in the periphery of the vesicles dispersed in the cytoplasm in TEM. Investigation of the labeling mechanism by inhibiting different uptake pathways revealed that endocytosis via scavenger receptor A, a process known to be clathrin mediated, plays a central role in the cellular uptake kinetics of both macrophages subpopulations. Biocompatibility evaluation showed no variation in cell viability and mitochondrial membrane potential with a low release of ROS. Flow cytometry and measurement of iNOS and Arginase 1 activity as marker of M1 and M2 macrophages polarization confirmed that magnetic labeling of macrophages subsets did not affect their polarization. In addition, no variation was observed in the biodistribution of magnetic iron oxide-labeled M1 and M2 macrophages subsets when monitored using noninvasive magnetic resonance imaging with a better detection for the enhanced SPIO–PEG–COOH-labeled cells.

SWCNTs as novel theranostic nanocarriers for cancer diagnosis and therapy: towards safe translation to the clinics

With their unique physicochemical properties, single walled carbon nanotubes (SWCNTs) hold great promise for applications as drug delivery systems (DDS) for early and better diagnosis and therapy of cancer. While several in vitro and in vivo studies have validated their potential benefit, no SWCNT-based formulation has yet reached clinical trials. Towards prospective safe clinical applications, the main properties that were adopted to enhance the biocompatibility of SWCNTs were highlighted. Then, the recent progresses in the in vivo applications of SWCNTs as diagnostic nanoprobes using multimodality imaging techniques and as therapeutic nanocarriers delivering wide range of anticancer efficient drugs to tumors were reviewed. Finally, the efforts required for safe clinical applications of SWCNTs as DDS for cancer diagnosis and therapy were discussed.