Functionalized superparamagnetic iron oxide nanoparticles provide highly efficient iron-labelling in macrophages for magnetic resonance-based detection in vivo

Tracking cells during regenerative cytotherapy is crucial for monitoring their safety and efficacy. Macrophages are an emerging cell-based regenerative therapy for liver disease and can be readily labeled for medical imaging. A reliable, clinically applicable cell-tracking agent would be a powerful tool to study cell biodistribution.Using a recently described chemical design, we set out to functionalize, optimize and characterize a new set of superparamagnetic iron oxide nanoparticles (SPIONs) to efficiently label macrophages for magnetic resonance imaging-based cell tracking in vivo.A series of cell health and iron uptake assays determined that positively charged SPIONs (+16.8 mV) could safely label macrophages more efficiently than the formerly approved ferumoxide (-6.7 mV; Endorem) and at least 10 times more efficiently than the clinically approved SPION ferumoxytol (-24.2 mV; Rienso). An optimal labeling time of 4 h at 25 µg/mL was demonstrated to label macrophages of mouse and human origin without any adverse effects on cell viability whilst providing substantial iron uptake (>5 pg Fe/cell) that was retained for 7 days in vitro. SPION labeling caused no significant reduction in phagocytic activity and a shift toward a reversible M1-like phenotype in bone marrow-derived macrophages (BMDMs). Finally, we show that SPION-labeled BMDMs delivered via the hepatic portal vein to mice are localized in the hepatic parenchyma resulting in a 50% drop in T2* in the liver. Engraftment of exogenous cells was confirmed via immunohistochemistry up to 3 weeks posttransplantation.A positively charged dextran-coated SPION is a promising tool to noninvasively track hepatic macrophage localization for therapeutic monitoring

Improved dynamic response assessment for intra-articular injected iron oxide nanoparticles

The emerging importance of nanoparticle technology, including iron oxide nanoparticles for monitoring development, progression, and treatment of inflammatory diseases such as arthritis, drives development of imaging techniques. Studies require an imaging protocol that is sensitive and quantifiable for the detection of iron oxide over a wide range of concentrations. Conventional signal loss measurements of iron oxide nanoparticle containing tissues saturate at medium concentrations and show a nonlinear/nonproportional intensity to concentration profile due to the competing effects of T1 and T2 relaxation. A concentration calibration phantom and an in vivo study of intra-articular injection in a rat knee of known concentrations of iron oxide were assessed using the difference-ultrashort echo time sequence giving a positive, quantifiable, unambiguous iron signal and monotonic, increasing concentration response over a wide concentration range in the phantom with limited susceptibility artifacts and high contrast in vivo to all other tissues. This improved dynamic response to concentration opens possibilities for quantification due to its linear nature at physiologically relevant concentrations

Monitoring the effects of dexamethasone treatment by MRI using in vivo iron oxide nanoparticle-labeled macrophages

Contains fulltext : 137472.pdf (publisher's version ) (Open Access)INTRODUCTION: Rheumatoid arthritis (RA) is a chronic disease causing recurring inflammatory joint attacks. These attacks are characterized by macrophage infiltration contributing to joint destruction. Studies have shown that RA treatment efficacy is correlated to synovial macrophage number. The aim of this study was to experimentally validate the use of in vivo superparamagnetic iron oxide nanoparticle (SPION) labeled macrophages to evaluate RA treatment by MRI. METHODS: The evolution of macrophages was monitored with and without dexamethasone (Dexa) treatment in rats. Two doses of 3 and 1 mg/kg Dexa were administered two and five days following induction of antigen induced arthritis. SPIONs (7 mg Fe/rat) were injected intravenously and the knees were imaged in vivo on days 6, 10 and 13. The MR images were scored for three parameters: SPION signal intensity, SPION distribution pattern and synovial oedema. Using 3D semi-automated software, the MR SPION signal was quantified. The efficacy of SPIONs and gadolinium chelate (Gd), an MR contrast agent, in illustrating treatment effects were compared. Those results were confirmed through histological measurements of number and area of macrophages and nanoparticle clusters using CD68 immunostaining and Prussian blue staining respectively. Results : Results show that the pattern and the intensity of SPION-labeled macrophages on MRI were altered by Dexa treatment. While the Dexa group had a uniform elliptical line surrounding an oedema pocket, the untreated group showed a diffused SPION distribution on day 6 post-induction. Dexa reduced the intensity of SPION signal 50-60% on days 10 and 13 compared to controls (P = 0.00008 and 0.002 respectively). Similar results were found when the signal was measured by the 3D tool. On day 13, the persisting low grade arthritis progression could not be demonstrated by Gd. Analysis of knee samples by Prussian blue and CD68 immunostaining confirmed in vivo SPION uptake by macrophages. Furthermore, CD68 immunostaining revealed that Dexa treatment significantly decreased the area and number of synovial macrophages. Prussian blue quantification corresponded to the macrophage measurements and both were in agreement with the MRI findings. CONCLUSIONS: We have demonstrated the feasibility of MRI tracking of in vivo SPION-labeled macrophages to assess RA treatment effects

Elucidating the Role if Integrin-extracellular Matrix Protein Interactions in Regulating Osteoclast Activity

Millions of people around the world suffer from the debilitating effects of inflammatory bone diseases characterized by excessive bone loss due to an increase in osteoclast formation and activity. Osteoclasts are multinucleated cells responsible for bone resorption in health and disease. Arthritic joints also have elevated levels of extracellular matrix proteins affecting the disease progression. The interaction between osteoclasts and the external milieu comprised of extracellular matrix proteins through integrins is essential for modulating the formation and activity of osteoclasts. The focus of this thesis was to elucidate how the interaction between the extracellular matrix proteins and osteoclasts regulates osteoclast formation and activity and the role of alphavbeta3 in this process. In primary rabbit osteoclast cultures, blocking the integrin alphavbeta3 using Vitaxin, an anti-human alphavbeta3 antibody, decreased osteoclast resorption by decreasing osteoclast attachment. Vitaxin’s inhibitory effect on osteoclast attachment was enhanced when osteoclasts were pretreated with M-CSF, a growth factor known to induce an activated conformation of the integrin alphavbeta3. Using the RAW264.7 cell line, the effects of the matrix proteins fibronectin and vitronectin on osteoclast activity were compared to those of osteopontin. Both fibronectin and vitronectin decreased the number of osteoclasts formed compared to osteopontin. Fibronectin’s effect on osteoclastogenesis was through decreasing pre-osteoclast migration and/or fusion but not through inhibiting their recruitment. In contrast, fibronectin induced resorption through increasing resorptive activity per osteoclast in comparison to vitronectin and osteopontin. These stimulatory effects were accompanied by an increase in the pro-inflammatory cytokines nitric oxide and IL-1beta Crosstalk between the signalling pathways of nitric oxide and IL-1betawas suggested by the ability of the nitric oxide inhibitor to decrease the level of IL-1beta which occurred exclusively on fibronectin. Osteoclasts on fibronectin also had a compact morphology with the smallest planar area while vitronectin increased the percentage of osteoclast with migratory morphology and osteopontin induced osteoclast spreading. The increase in compact morphology on fibronectin was associated with a decrease in extracellular pH. Low extracellular pH was found to increase the total time osteoclasts spend in a compact phase. These results show that matrix proteins differentially regulate osteoclast formation, activity and morphology.Ph

Elucidating the Role if Integrin-extracellular Matrix Protein Interactions in Regulating Osteoclast Activity

Millions of people around the world suffer from the debilitating effects of inflammatory bone diseases characterized by excessive bone loss due to an increase in osteoclast formation and activity. Osteoclasts are multinucleated cells responsible for bone resorption in health and disease. Arthritic joints also have elevated levels of extracellular matrix proteins affecting the disease progression. The interaction between osteoclasts and the external milieu comprised of extracellular matrix proteins through integrins is essential for modulating the formation and activity of osteoclasts. The focus of this thesis was to elucidate how the interaction between the extracellular matrix proteins and osteoclasts regulates osteoclast formation and activity and the role of alphavbeta3 in this process. In primary rabbit osteoclast cultures, blocking the integrin alphavbeta3 using Vitaxin, an anti-human alphavbeta3 antibody, decreased osteoclast resorption by decreasing osteoclast attachment. Vitaxin’s inhibitory effect on osteoclast attachment was enhanced when osteoclasts were pretreated with M-CSF, a growth factor known to induce an activated conformation of the integrin alphavbeta3. Using the RAW264.7 cell line, the effects of the matrix proteins fibronectin and vitronectin on osteoclast activity were compared to those of osteopontin. Both fibronectin and vitronectin decreased the number of osteoclasts formed compared to osteopontin. Fibronectin’s effect on osteoclastogenesis was through decreasing pre-osteoclast migration and/or fusion but not through inhibiting their recruitment. In contrast, fibronectin induced resorption through increasing resorptive activity per osteoclast in comparison to vitronectin and osteopontin. These stimulatory effects were accompanied by an increase in the pro-inflammatory cytokines nitric oxide and IL-1beta Crosstalk between the signalling pathways of nitric oxide and IL-1betawas suggested by the ability of the nitric oxide inhibitor to decrease the level of IL-1beta which occurred exclusively on fibronectin. Osteoclasts on fibronectin also had a compact morphology with the smallest planar area while vitronectin increased the percentage of osteoclast with migratory morphology and osteopontin induced osteoclast spreading. The increase in compact morphology on fibronectin was associated with a decrease in extracellular pH. Low extracellular pH was found to increase the total time osteoclasts spend in a compact phase. These results show that matrix proteins differentially regulate osteoclast formation, activity and morphology.Ph

A fast and reproducible method to quantify magnetic nanoparticle biodistribution

The quantification of nanoparticles, particularly superparamagnetic iron oxide nanoparticles (SPIONs), both in vitro and in vivo has become highly important in recent years. Some methods, such as induced coupled plasma (ICP) spectroscopy and UV-visible chemical titration using Prussian Blue (PB), already exist however they consist of the titration of the whole iron content. These standard methods need sample preparations leading to their destruction and long measurement time. In this study, we used magnetic susceptibility measurements (MSM) to titrate the concentration and biodistribution of magnetic particles in the organs of rats. The advantages of the MSM SPION quantification technique are presented and compared to widely used methods of iron oxide titration such as ICP and PB UV-visible titration. We have demonstrated that MSM is a simpler, faster (1 second per measurement), more reproducible and highly sensitive technique for SPION detection with minimal detection around 2 μgFe mL(-1) without being influenced by neither the SPION coating nor their surrounding environment. Moreover, MSM is a more robust method as it is not affected by endogenous iron facilitating the distinction of SPIONs (iron present as nanoparticles) from background iron in tissues. This advantage allows the decrease of control samples needed in biological studies. In conclusion, we have demonstrated that MSM is a standard method that can be easily setup to determine the biodistribution of SPIONs regardless of their environment