LaPO4 nanoparticles doped with actinium-225 that partially sequester daughter radionuclides

Nanoscale materials have been envisioned as carriers for various therapeutic drugs, including radioisotopes. Inorganic nanoparticles (NPs) are particularly appealing vehicles for targeted radiotherapy because they can package several radioactive atoms into a single carrier and can potentially retain daughter radioisotopes produced by in vivo generators such as actinium-225 ((225)Ac, t(1/2) = 10 d). Decay of this radioisotope to stable bismuth-209 proceeds through a chain of short-lived daughters accompanied by the emission of four α-particles that release >27 MeV of energy. The challenge in realizing the enhanced cytotoxic potential of in vivo generators lies in retaining the daughter nuclei at the therapy site. When (225)Ac is attached to targeting agents via standard chelate conjugation methods, all of the daughter radionuclides are released after the initial α-decay occurs. In this work, (225)Ac was incorporated into lanthanum phosphate NPs to determine whether the radioisotope and its daughters would be retained within the dense mineral lattice. Further, the (225)Ac-doped NPs were conjugated to the monoclonal antibody mAb 201B, which targets mouse lung endothelium through the vasculature, to ascertain the targeting efficacy and in vivo retention of radioisotopes. Standard biodistribution techniques and microSPECT/CT imaging of (225)Ac as well as the daughter radioisotopes showed that the NPs accumulated rapidly in mouse lung after intravenous injection. By showing that excess, competing, uncoupled antibodies or NPs coupled to control mAbs are deposited primarily in the liver and spleen, specific targeting of NP-mAb 201B conjugates was demonstrated. Biodistribution analysis showed that ～30% of the total injected dose of La((225)Ac)PO(4) NPs accumulated in mouse lungs 1 h postinjection, yielding a value of % ID/g >200. Furthermore, after 24 h, 80% of the (213)Bi daughter produced from (225)Ac decay was retained within the target organ and (213)Bi retention increased to ～87% at 120 h. In vitro analyses, conducted over a 1 month interval, demonstrated that ～50% of the daughters were retained within the La((225)Ac)PO(4) NPs at any point over that time frame. Although most of the γ-rays from radionuclides in the (225)Ac decay chain are too energetic to be captured efficiently by SPECT detectors, appropriate energy windows were found that provided dramatic microSPECT images of the NP distribution in vivo. We conclude that La((225)Ac)PO(4)-mAb 201B conjugates can be targeted efficiently to mouse lung while partially retaining daughter products and that targeting can be monitored by biodistribution techniques and microSPECT imaging.     

Synthesis of tumor-targeted folate conjugated fluorescent magnetic albumin nanoparticles for enhanced intracellular dual-modal imaging into human brain tumor cells

Superparamagnetic iron oxide nanoparticles (SPIO NPs), utilized as carriers are attractive materials widely applied in biomedical fields, but target-specific SPIO NPs with lower toxicity and excellent biocompatibility are still lacking for intracellular visualization in human brain tumor diagnosis and therapy. Herein, bovine serum albumin (BSA) coated superparamagnetic iron oxide, i.e. γ-Fe₂O₃ nanoparticles (BSA-SPIO NPs), are synthesized. Tumor-specific ligand folic acid (FA) is then conjugated onto BSA-SPIO NPs to fabricate tumor-targeted NPs, FA-BSA-SPIO NPs as a contrast agent for MRI imaging. The FA-BSA-SPIO NPs are also labeled with fluorescein isothiocyanate (FITC) for intracellular visualization after cellular uptake and internalization by glioma U251 cells. The biological effects of the FA-BSA-SPIO NPs are investigated in human brain tumor U251 cells in detail. These results show that the prepared FA-BSA-SPIO NPs display undetectable cytotoxicity, excellent biocompatibility, and potent cellular uptake. Moreover, the study shows that the made FA-BSA-SPIO NPs are effectively internalized for MRI imaging and intracellular visualization after FITC labeling in the targeted U251 cells. Therefore, the present study demonstrates that the fabricated FITC-FA-BSA-SPIO NPs hold promising perspectives by providing a dual-modal imaging as non-toxic and target-specific vehicles in human brain tumor treatment in future.     

Adjusting the Balance between Effective Loading and Vector Migration of Macrophage Vehicles to Deliver Nanoparticles

The nature of macrophage allows the possibility that this cell type could be used as drug delivery system to track therapeutic drug nanoparticles (NPs) in cancer. However, there is no existing research on the regulation between effective loading of NPs and targeted delivery of macrophages. Here, we investigated the important parameters of intracellular NP quantity and the vector migration rate. Macrophage loading capacity was obtained by comparing the uptake quantity of varisized NPs, and the delivery ability of loaded cells was determined by measuring vector migration rates. We observed a positive correlation between the size of NPs and directed macrophage migration. Our findings suggest that the molecular mechanism of migration vector rate regulation involved increased expression levels of colony-stimulating factor-1 (CSF-1) receptor and integrin induced by 100-nm and 500-nm particles. The ability of macrophages uptake to varisized NPs showed the opposite trend, with the increased vector rate of cell migration influenced by NPs. We are able to demonstrate the important balance between effective macrophage loading and targeted delivery. By adjusting the balance parameters, it will be possible to utilize NPs in macrophage-mediated disease diagnosis and therapy.     

Glypican-3 targeting of liver cancer cells using multifunctional nanoparticles

Imaging is essential in accurately detecting, staging, and treating primary liver cancer (hepatocellular carcinoma [HCC]), one of the most prevalent and lethal malignancies. We developed a novel multifunctional nanoparticle (NP) specifically targeting glypican-3 (GPC3), a proteoglycan implicated in promotion of cell growth that is overexpressed in most HCCs. Quantitative real-time polymerase chain reaction was performed to confirm the differential GPC3 expression in two human HCC cells, Hep G2 (high) and HLF (negligible). These cells were treated with biotin-conjugated GPC3 monoclonal antibody (αGPC3) and subsequently targeted using superparamagnetic iron oxide NPs conjugated to streptavidin and Alexa Fluor 647. Flow cytometry demonstrated that only GPC3-expressing Hep G2 cells were specifically targeted using this αGPC3-NP conjugate (fourfold mean fluorescence over nontargeted NP), and magnetic resonance imaging (MRI) experiments showed similar findings (threefold R2 relaxivity). Confocal fluorescence microscopy localized the αGPC3 NPs only to the cell surface of GPC3-expressing Hep G2 cells. Further characterization of this construct demonstrated a negatively charged, monodisperse, 50 nm NP, ideally suited for tumor targeting. This GPC3-specific NP system, with dual-modality imaging capability, may enhance pretreatment MRI, enable refined intraoperative HCC visualization by near-infrared fluorescence, and be potentially used as a carrier for delivery of tumor-targeted therapies, improving patient outcomes.     

Targeted gene delivery to CD117‐expressing cells in vivo with lentiviral vectors co‐displaying stem cell factor and a fusogenic molecule

The development of a lentiviral system to deliver genes to specific cell types could improve the safety and the efficacy of gene delivery. Previously, we have developed an efficient method to target lentivectors to specific cells via an antibody–antigen interaction in vitro and in vivo. We report herein a targeted lentivector that harnesses the natural ligand–receptor recognition mechanism for targeted modification of c‐KIT receptor‐expressing cells. For targeting, we incorporate membrane‐bound human stem cell factor (hSCF), and for fusion, a Sindbis virus‐derived fusogenic molecule (FM) onto the lentiviral surface. These engineered vectors can recognize cells expressing surface CD117, resulting in efficient targeted transduction of cells in an SCF‐receptor dependent manner in vitro, and in vivo in xenografted mouse models. This study expands the ability of targeting lentivectors beyond antibody targets to include cell‐specific surface receptors. Development of a high titer lentivector to receptor‐specific cells is an attractive approach to restrict gene expression and could potentially ensure therapeutic effects in the desired cells while limiting side effects caused by gene expression in non‐target cells. Biotechnol. Bioeng. 2009; 104: 206–215 © 2009 Wiley Periodicals, Inc.     

Improved targeting of ligand-modified adenovirus as a new near infrared fluorescence tumor imaging probe

E1/E3-deleted Adenovirus 5 (Ad.5) possesses a great potential in gene therapy because of its high efficacy in gene transfer and low toxicity. Studies have shown that Coxsackie-Adenovirus receptor (CAR) is the determinant factor for the targeting of Adenovirus vectors. To extend the natural targeting of Ad to low CAR expressing tumors, we covalently attached folic acid (FA) to E1/E3-deleted Ad.5 capsids. Near-infrared (NIR) fluorescent dye ICG-Der-02 was subsequently conjugated with FA-Ad particles for in vivo imaging. The cell experiments and acute toxicity studies demonstrated the low toxicity of FA-Ad-ICG02 to normal cell/tissues. The dynamic behavior and targeting ability of FA-Ad-ICG02 to different tumors were investigated by NIR fluorescence imaging. In vitro and in vivo studies demonstrated its high targeting capability to CAR or FR positive tumors. The results support the potential of using ligand-modified Ad probe for tumor diagnosis and targeted therapy.     

Synthesis of fluorescent and low cytotoxicity phenol formaldehyde resin (PFR)@Ag composites for cell imaging and antibacterial activity

Ag nanoparticles (NPs) were loaded onto the surface of phenol formaldehyde resin (PFR) NPs without any reducing agent. The as‐synthesized PFR@Ag composites have low cytotoxicity, which makes them promising antibacterial agents. Furthermore, the good fluorescence of PFR could be used for cell imaging. Copyright © 2015 John Wiley & Sons, Ltd.     

Cellular MR imaging

Cellular MR imaging is a young field that aims to visualize targeted cells in living organisms. In order to provide a different signal intensity of the targeted cell, they are either labeled with MR contrast agents in vivo or prelabeled in vitro. Either (ultrasmall) superparamagnetic iron oxide [(U)SPIO] particles or (polymeric) paramagnetic chelates can be used for this purpose. For in vivo cellular labeling, Gd3+- and Mn2+-chelates have mainly been used for targeted hepatobiliary imaging, and (U)SPIO-based cellular imaging has been focused on imaging of macrophage activity. Several of these magnetopharmaceuticals have been FDA-approved or are in late-phase clinical trials. As for prelabeling of cells in vitro, a challenge has been to induce a sufficient uptake of contrast agents into nonphagocytic cells, without affecting normal cellular function. It appears that this issue has now largely been resolved, leading to an active research on monitoring the cellular biodistribution in vivo following transplantation or transfusion of these cells, including cell migration and trafficking. New applications of cellular MR imaging will be directed, for instance, towards our understanding of hematopoietic (immune) cell trafficking and of novel guided (stem) cell-based therapies aimed to be translated to the clinic in the future.     

Antigen itself antibody: an alternative one‐step immunoassay for measuring the anti‐idiotypic antibody titer

Immunoassay designs rely on the great specificity of antibodies and a suitable marker that facilitates generation of a quantitative signal. Currently, there is no reliable method for measuring the titers of an anti‐idiotypic antibody. Our initial attempt to measure titers of mouse anti‐idiotypic antibody after idiotypic vaccination with HM‐1 killer toxin neutralizing monoclonal antibody (nmAb‐KT) failed. Because the injected antigen, nmAb‐KT, is a mouse IgG, using a commercial antibody to measure the antibody titer always gave a false positive signal against control mouse serum antibody in parallel with the antigen‐treated immunized serum antibodies. To get a reliable and clearly differentiable signal by ELISA, idiotypic antigen was labeled with HRP and HRP‐conjugated‐nmAb‐KT used to measure the antibody titers in the antigen‐treated mice. Compared with control mice, signals were found in high anti‐nmAb‐KT IgG responses in test mice; however, untreated control mice had a significant amount of purified non‐specific IgG. This method is amenable to long read lengths and will likely enable anti‐idiotypic antibody titer measurement in a more specific and cost effective way without requiring commercial antibody.     

Targeting of MPEG-protected polyamino acid carrier to human E-selectin in vitro

Summary.  Targeted diagnostic agents are expected to have a significant impact in molecular imaging of cell-surface associated markers of proliferation, inflammation and angiogenesis. In this communication, we describe a new class of targeted polyamino acid-based protected graft copolymers (PGC) of poly-(L-lysine) and methyl poly-(ethylene glycol) (PGC) covalently conjugated with a monoclonal antibody fragment, F(ab′)₂. We utilized targeted PGC conjugates as carriers of near-infrared indocyanine fluorophores (Cy5.5) for optical imaging of endothelial cell populations expressing IL-1β inducible proinflammatory marker E-selectin. We compared two conjugation chemistries, involving either introduction of sulfhydryl group to F(ab′)₂, or via direct attachment of the antibody fragment directly to the chemically activated PGC. Both PGC-based targeted agents demonstrated high binding specificity (20–30 fold over non-specific uptake) and were utilized for imaging E-selectin expression on human endothelial cells activated with IL-1β. Received June 29, 2001 Accepted August 8, 2001 Published online August 9, 2002     

Characterization of antilytic peptide antibody: application for the detection of lytic‐based hybrid peptide in serum samples

We previously reported that a novel targeted drug termed hybrid epidermal growth factor receptor (EGFR)‐lytic peptide, made by chemical conjugation of targeted binding peptide and cell‐killing, lytic‐peptide components, has selective cytotoxic activity that allows it to discriminate between normal and cancer cells. In addition, in vivo analysis revealed that this hybrid peptide displays significant antitumor activity in a xenograft model of human breast and pancreatic cancer in mice. Here, we characterized antilytic peptide antibody, which was raised from rabbit serum using the antigen of lytic peptide conjugated with keyhole limpet hemocyanin. It was found that antilytic peptide antibody is specific to the lytic peptide as assessed by both ELISA and surface plasmon resonance analysis and can also bind to EGFR‐lytic peptide. Epitope mapping analysis using Biacore showed that two successive lysine regions in the lytic‐peptide sequence are significant for recognition by this antibody. In addition, it was shown that this antibody can detect lytic‐based hybrid peptide in serum samples from mouse blood and also in cultured breast cancer MDA‐MB‐231 cell samples by immunocytochemical staining experiments. It was found that the maximum concentrations of this peptide in serum were reached within 15–30 min of i.v. administration of EGFR‐lytic peptide to mice. These results indicate that this antibody will be a useful tool for the detection of lytic‐based peptides to investigate their in vivo stability and pharmacokinetics. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Tumor-targeted pH-low insertion peptide delivery of theranostic gadolinium nanoparticles for image-guided nanoparticle-enhanced radiation therapy

Tumor targeting studies using metallic nanoparticles (NPs) have shown that the enhanced permeability and retention effect may not be sufficient to deliver the amount of intratumoral and intracellular NPs needed for effective in vivo radiosensitization. This work describes a pH-Low Insertion Peptide (pHLIP) targeted theranostic agent to enable image-guided NP-enhanced radiotherapy using a clinically feasible amount of injected NPs. Conventional gadolinium (Gd) NPs were conjugated to pHLIPs and evaluated in vitro for radiosensitivity and in vivo for mouse MRI. Cultured A549 human lung cancer cells were incubated with 0.5 mM of pHLIP-GdNP or conventional GdNP. Mass spectrometry showed 78-fold more cellular Gd uptake with pHLIP-GdNPs, and clonogenic survival assays showed 44% more enhanced radiosensitivity by 5 Gy irradiation with pHLIP-GdNPs at pH 6.2. In contrast to conventional GdNPs, MR imaging of tumor-bearing mice showed pHLIP-GdNPs had a long retention time in the tumor (>9 h), suitable for radiotherapy, and penetrated into the poorly-vascularized tumor core. The Gd-enhanced tumor corresponded with low-pH areas also independently measured by an in vivo molecular MRI technique. pHLIPs actively target cell surface acidity from tumor cell metabolism and deliver GdNPs into cells in solid tumors. Intracellular delivery enhances the effect of short-range radiosensitizing photoelectrons and Auger electrons. Because acidity is a general hallmark of tumor cells, the delivery is more general than antibody targeting. Imaging the in vivo NP biodistribution and more acidic (often more aggressive) tumors has the potential for quantitative radiotherapy treatment planning and pre-selecting patients who will likely benefit more from NP radiation enhancement.     

Synthesis and radiolabeling of chelator-RNA aptamer bioconjugates with copper-64 for targeted molecular imaging

Ribonucleic acid (RNA) aptamers with high affinity and specificity for cancer-specific cell-surface antigens are promising reagents for targeted molecular imaging of cancer using positron emission tomography (PET). For this application, aptamers must be conjugated to chelators capable of coordinating PET-radionuclides (e.g., copper-64, (64)Cu) to enable radiolabeling for in vivo imaging of tumors. This study investigates the choice of chelator and radiolabeling parameters such as pH and temperature for the development of (64)Cu-labeled RNA-based targeted agents for PET imaging. The characterization and optimization of labeling conditions are described for four chelator-aptamer complexes. Three commercially available bifunctional macrocyclic chelators (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid mono N-hydroxysuccinimide [DOTA-NHS]; S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid [p-SCN-Bn-NOTA]; and p-SCN-Bn-3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid [p-SCN-Bn-PCTA]), as well as the polyamino-macrocyclic diAmSar (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane-1,8-diamine) were conjugated to A10-3.2, a RNA aptamer which has been shown to bind specifically to a prostate cancer-specific cell-surface antigen (PSMA). Although a commercial bifunctional version of diAmSar was not available, RNA conjugation with this chelator was achieved in a two-step reaction by the addition of a disuccinimidyl suberate linker. Radiolabeling parameters (e.g., pH, temperature, and time) for each chelator-RNA conjugate were assessed in order to optimize specific activity and RNA stability. Furthermore, the radiolabeled chelator-coupled RNA aptamers were evaluated for binding specificity to their target antigen. In summary, key parameters were established for optimal radiolabeling of RNA aptamers for eventual PET imaging with (64)Cu.     

AS1411 aptamer tagged PLGA-lecithin-PEG nanoparticles for tumor cell targeting and drug delivery

Liposomes and polymers are widely used drug carriers for controlled release since they offer many advantages like increased treatment effectiveness, reduced toxicity and are of biodegradable nature. In this work, anticancer drug‐loaded PLGA‐lecithin‐PEG nanoparticles (NPs) were synthesized and were functionalized with AS1411 anti‐nucleolin aptamers for site‐specific targeting against tumor cells which over expresses nucleolin receptors. The particles were characterized by transmission electron microscope (TEM) and X‐ray photoelectron spectroscopy (XPS). The drug‐loading efficiency, encapsulation efficiency and in vitro drug release studies were conducted using UV spectroscopy. Cytotoxicity studies were carried out in two different cancer cell lines, MCF‐7 and GI‐1 cells and two different normal cells, L929 cells and HMEC cells. Confocal microscopy and flowcytometry confirmed the cellular uptake of particles and targeted drug delivery. The morphology analysis of the NPs proved that the particles were smooth and spherical in shape with a size ranging from 60 to 110 nm. Drug‐loading studies indicated that under the same drug loading, the aptamer‐targeted NPs show enhanced cancer killing effect compared to the corresponding non‐targeted NPs. In addition, the PLGA‐lecithin‐PEG NPs exhibited high encapsulation efficiency and superior sustained drug release than the drug loaded in plain PLGA NPs. The results confirmed that AS1411 aptamer‐PLGA‐lecithin‐PEG NPs are potential carrier candidates for differential targeted drug delivery. Biotechnol. Bioeng. 2012; 109: 2920–2931. © 2012 Wiley Periodicals, Inc.     

Targeted antigen presentation using crosslinked antibody heteroaggregates

We have targeted protein antigens to antigen-presenting cells in vitro by using antibody heteroaggregates containing an antibody against a protein antigen covalently crosslinked to an antibody against a target structure on the surface of the antigen-presenting cells. Antigen presentation was assessed by measurement of lymphokine released by antigen-specific T cell hybridomas. Depending on the experimental conditions, the crosslinked antibodies decreased the amount of antigen required to give a response by the hybridomas by factors of 10(2) to 10(3). Enhanced presentation occurred when antigen was targeted to MHC class I and class II molecules, surface immunoglobulin, or Fc gamma receptors on the surface of the murine B cell lymphoma-hybridoma, TA3. An enhancement of antigen presentation also occurred when antigen was targeted to surface IgD, or class I and class II MHC molecules on murine splenic B cells, and when antigen was targeted to class I and class II molecules on irradiated adherent spleen cells. No response was seen when antigen was targeted to Fc gamma R on B cells or adherent spleen cells. The ability of each crosslinked antibody to enhance presentation paralleled the total amount of each that bound to the surface of the antigen-presenting cells. Antigen presentation, mediated by crosslinked antibody, was antigen-specific and I-A restricted. The presentation of one antigen by using crosslinked antibody did not result in enhanced presentation of a second, bystander antigen. These results suggest that a novel means of stimulating immune responses may be possible in vivo, by targeting antigen to surface structures on antigen-presenting cells.     

Sulfonates-PMMA nanoparticles conjugates: A versatile system for multimodal application

We report herein the viability of a novel nanoparticles (NPs) conjugated system, namely the attachment, based on ionic and hydrophobic interactions, of different sulfonated organic salts to positively charged poly(methylmethacrylate) (PMMA)-based core-shell nanoparticles (EA0) having an high density of ammonium groups on their shells. In this context three different applications of the sulfonates@EA0 systems have been described. In detail, their ability as cytotoxic drugs and pro-drugs carriers was evaluated in vitro on NCI-H460 cell line and in vivo against human ovarian carcinoma IGROV-1 cells. Besides, 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) was chosen for NPs loading, and its internalization as bioimaging probe was evaluated on Hep G2 cells. Overall, the available data support the interest for these PMMA NPs@sulfonates systems as a promising formulation for theranostic applications. In vivo biological data strongly support the potential value of these core-shell NPs as delivery system for negatively charged drugs or biologically active molecules. Additionally, we have demonstrated the ability of these PMMA core-shell nanoparticles to act as efficient carriers of fluorophores. In principle, thanks to the high PMMA NPs external charge density, sequential and very easy post-loading of different sulfonates is achievable, thus allowing the preparation of nanocarriers either with bi-modal drug delivery behaviour or as theranostic systems.     

Internalization of paramagnetic phosphatidylserine-containing liposomes by macrophages

ABSTRACT: BACKGROUND: Inflammation plays an important role in many pathologies, including cardiovascular diseases, neurological conditions and oncology, and is considered an important predictor for disease progression and outcome. In vivo imaging of inflammatory cells will improve diagnosis and provide a read-out for therapy efficacy. Paramagnetic phosphatidylserine (PS)-containing liposomes were developed for magnetic resonance imaging (MRI) and confocal microscopy imaging of macrophages. These nanoparticles also provide a platform to combine imaging with targeted drug delivery. RESULTS: Incorporation of PS into liposomes did not affect liposomal size and morphology up to 12 mol% of PS. Liposomes containing 6 mol% of PS showed the highest uptake by murine macrophages, while only minor uptake was observed in endothelial cells. Uptake of liposomes containing 6 mol% of PS was dependent on the presence of Ca2+ and Mg2+. Furthermore, these 6 mol% PS-containing liposomes were mainly internalized into macrophages, whereas liposomes without PS only bound to the macrophage cell membrane. CONCLUSIONS: Paramagnetic liposomes containing 6 mol% of PS for MR imaging of macrophages have been developed. In vitro these liposomes showed specific internalization by macrophages. Therefore, these liposomes might be suitable for in vivo visualization of macrophage content and for (visualization of) targeted drug delivery to inflammatory cells.     

Hyperspectral darkfield microscopy of PEGylated gold nanoparticles targeting CD44‐expressing cancer cells

We present a new hyperspectral darkfield imaging system with a scanned broadband supercontinuum light source. We observed the specific attachment of the functionalized gold plasmonic nanoparticles (AuNPs) targeting CD44⁺ human breast cancer cells by conventional and by proposed hyperspectral darkfield microscopy. This wide‐field and low phototoxic hyperspectral imaging system has been successful for performing spectral three‐dimensional (3D) localization and spectroscopic identification of CD44‐targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic‐based imaging, disease detection and treatment in complex biological environment. Presented system capability for 3D NP tracking will also enable investigation of specific sub‐cellular activity with the use of NPs as spectral sensors. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Imagerie moléculaire de la plaque d’athérome vulnérable. Évaluation préclinique et clinique de traceurs radioactifs

Atherosclerotic cardiovascular diseases (CVD) are the leading cause of mortality worldwide, accounting for greater than 19.10⁶ deaths annually. Despite major advances in the treatment of CVD, a high proportion of CVD victims die suddenly while being apparently healthy, the great majority of these accidents being due to the rupture or erosion of a vulnerable coronary atherosclerotic plaque. Indeed, an acute heart attack is the first symptom of atherosclerosis in as much as 50% of individuals with severe disease. A non-invasive imaging methodology allowing the early detection of vulnerable atherosclerosis in selected individuals prior to the occurrence of any symptom would therefore be of great public health benefit. Nuclear imaging could potentially allow the identification of vulnerable patients by non-invasive scintigraphic imaging following administration of a radiolabeled tracer. The development of radiolabeled probes that specifically bind to and allow the in vivo imaging of vulnerable atherosclerotic plaques is therefore the subject of intense ongoing experimental and clinical research. Radiotracers targeted at the inflammatory process seem particularly relevant and promising. Recently, macrophage targeting allowed the experimental in vivo detection of atherosclerosis using either SPECT or PET imaging. A few tracers have also been evaluated clinically. Targeting of apoptosis and macrophage metabolism both allowed the imaging of vulnerable atherosclerotic plaques in the carotid vessels of patients. However, nuclear imaging of vulnerable plaques at the level of the coronary arteries remains a challenging issue because of the small size of atherosclerotic lesions and of their vicinity with blood and the circulating tracer activity.     

The Uptake of Soluble and Particulate Antigens by Epithelial Cells in the Mouse Small Intestine

Intestinal epithelial cells (IECs) overlying the villi play a prominent role in absorption of digested nutrients and establish a barrier that separates the internal milieu from potentially harmful microbial antigens. Several mechanisms by which antigens of dietary and microbial origin enter the body have been identified; however whether IECs play a role in antigen uptake is not known. Using in vivo imaging of the mouse small intestine, we investigated whether epithelial cells (enterocytes) play an active role in the uptake (sampling) of lumen antigens. We found that small molecular weight antigens such as chicken ovalbumin, dextran, and bacterial LPS enter the lamina propria, the loose connective tissue which lies beneath the epithelium via goblet cell associated passageways. However, epithelial cells overlying the villi can internalize particulate antigens such as bacterial cell debris and inert nanoparticles (NPs), which are then found co-localizing with the CD11c+ dendritic cells in the lamina propria. The extent of NP uptake by IECs depends on their size: 20–40 nm NPs are taken up readily, while NPs larger than 100 nm are taken up mainly by the epithelial cells overlying Peyer''s patches. Blocking NPs with small proteins or conjugating them with ovalbumin does not inhibit their uptake. However, the uptake of 40 nm NPs can be inhibited when they are administered with an endocytosis inhibitor (chlorpromazine). Delineating the mechanisms of antigen uptake in the gut is essential for understanding how tolerance and immunity to lumen antigens are generated, and for the development of mucosal vaccines and therapies.