Fluorescence labeled microbubbles for multimodal imaging.

Air-filled polyvinyl alcohol microbubbles (PVA-MBs) were recently introduced as a contrast agent for ultrasound imaging. In the present study, we explore the possibility of extending their application in multimodal imaging by labeling them with a near infrared (NIR) fluorophore, VivoTag-680. PVA-MBs were injected intravenously into FVB/N female mice and their dynamic biodistribution over 24 h was determined by 3D-fluorescence imaging co-registered with 3D-µCT imaging, to verify the anatomic location. To further confirm the biodistribution results from in vivo imaging, organs were removed and examined histologically using bright field and fluorescence microscopy. Fluorescence imaging detected PVA-MB accumulation in the lungs within the first 30 min post-injection. Redistribution to a low extent was observed in liver and kidneys at 4 h, and to a high extent mainly in the liver and spleen at 24 h. Histology confirmed PVA-MB localization in lung capillaries and macrophages. In the liver, they were associated with Kupffer cells; in the spleen, they were located mostly within the marginal-zone. Occasional MBs were observed in the kidney glomeruli and interstitium. The potential application of PVA-MBs as a contrast agent was also studied using ultrasound (US) imaging in subcutaneous and orthotopic pancreatic cancer mouse models, to visualize blood flow within the tumor mass. In conclusion, this study showed that PVA-MBs are useful as a contrast agent for multimodal imaging.

Flavin-containing monooxygenase 3 (FMO3) role in busulphan metabolic pathway.

Busulphan (Bu) is an alkylating agent used in the conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). Bu is extensively metabolized in the liver via conjugations with glutathione to form the intermediate metabolite (sulfonium ion) which subsequently is degraded to tetrahydrothiophene (THT). THT was reported to be oxidized forming THT-1-oxide that is further oxidized to sulfolane and finally 3-hydroxysulfolane. However, the underlying mechanisms for the formation of these metabolites remain poorly understood. In the present study, we performed in vitro and in vivo investigations to elucidate the involvement of flavin-containing monooxygenase-3 (FMO3) and cytochrome P450 enzymes (CYPs) in Bu metabolic pathway. Rapid clearance of THT was observed when incubated with human liver microsomes. Furthermore, among different recombinant microsomal enzymes, the highest intrinsic clearance for THT was obtained via FMO3 followed by several CYPs including 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4. In Bu- or THT-treated mice, inhibition of FMO3 by phenylthiourea significantly suppressed the clearance of both Bu and THT. Moreover, the simultaneous administration of a high dose of THT (200µmol/kg) to Bu-treated mice reduced the clearance of Bu. Consistently, in patients undergoing HSCT, repeated administration of Bu resulted in a significant up-regulation of FMO3 and glutathione-S-transfrase -1 (GSTA1) genes. Finally, in a Bu-treated patient, additional treatment with voriconazole (an antimycotic drug known as an FMO3-substrate) significantly altered the Bu clearance. In conclusion, we demonstrate for the first time that FMO3 along with CYPs contribute a major part in busulphan metabolic pathway and certainly can affect its kinetics. The present results have high clinical impact. Furthermore, these findings might be important for reducing the treatment-related toxicity of Bu, through avoiding interaction with other concomitant used drugs during conditioning and hence improving the clinical outcomes of HSCT.

Complement activation is involved in the hepatic injury caused by high-dose exposure of mice to perfluorooctanoic acid.

High-dose exposure of mice to perfluorooctanoate (PFOA) induces both hepatotoxicity and immunotoxicity. Here, we characterized the effects of 10-day dietary treatment with PFOA (0.002-0.02%, w/w) on the liver and complement system of male C57BL/6 mice. At all four doses, this compound caused hepatomegaly and reduced the serum level of triglycerides (an indicator for activation of the peroxisome proliferator-activated receptor-alpha (PPARα)). At the highest dose (0.02%, w/w), this hepatomegaly was associated with the hepatic injury, as reflected in increased activity of alanine aminotranferase (ALAT) in the serum, severe hepatocyte hypertrophy and hepatocellular necrosis. PFOA-induced hepatic injury was associated with in vivo activation of the complement system as indicated by (i) significant attenuation of the serum activities of both the classical and alternative pathways; (ii) a marked reduction in the serum level of the complement factor C3; and (iii) deposition of the complement factor C3 fragment (C3a) in the hepatic parenchyma. PFOA did not activate the alternative pathway of complement in vitro. At doses lower than 0.02%, PFOA induced hepatocyte hypertrophy without causing liver injury or activating complement. These results reveal substantial involvement of activation of complement in the pathogenesis of PFOA-induced hepatotoxicity.

Biodegradable polymeric vesicles containing magnetic nanoparticles, quantum dots and anticancer drugs for drug delivery and imaging.

We have developed biodegradable polymeric vesicles as a nanocarrier system for multimodal bio-imaging and anticancer drug delivery. The poly(lactic-co-glycolic acid) (PLGA) vesicles were fabricated by encapsulating inorganic imaging agents of superparamagnetic iron oxide nanoparticles (SPION), manganese-doped zinc sulfide (Mn:ZnS) quantum dots (QDs) and the anticancer drug busulfan into PLGA nanoparticles via an emulsion-evaporation method. T2∗-weighted magnetic resonance imaging (MRI) of PLGA-SPION-Mn:ZnS phantoms exhibited enhanced negative contrast with r2∗ relaxivity of approximately 523 s(-1) mM(-1) Fe. Murine macrophage (J774A) cellular uptake of PLGA vesicles started fluorescence imaging at 2 h and reached maximum intensity at 24 h incubation. The drug delivery ability of PLGA vesicles was demonstrated in vitro by release of busulfan. PLGA vesicle degradation was studied in vitro, showing that approximately 32% was degraded into lactic and glycolic acid over a period of 5 weeks. The biodistribution of PLGA vesicles was investigated in vivo by MRI in a rat model. Change of contrast in the liver could be visualized by MRI after 7 min and maximal signal loss detected after 4 h post-injection of PLGA vesicles. Histological studies showed that the presence of PLGA vesicles in organs was shifted from the lungs to the liver and spleen over time.

Biodistribution, kinetics, and biological fate of SPION microbubbles in the rat.

BACKGROUND: In the present investigation, we studied the kinetics and biodistribution of a contrast agent consisting of poly(vinyl alcohol) (PVA) microbubbles containing superparamagnetic iron oxide (SPION) trapped between the PVA layers (SPION microbubbles). METHODS: The biological fate of SPION microbubbles was determined in Sprague-Dawley rats after intravenous administration. Biodistribution and elimination of the microbubbles were studied in rats using magnetic resonance imaging for a period of 6 weeks. The rats were sacrificed and perfusion-fixated at different time points. The magnetic resonance imaging results obtained were compared with histopathologic findings in different organs. RESULTS: SPION microbubbles could be detected in the liver using magnetic resonance imaging as early as 10 minutes post injection. The maximum signal was detected between 24 hours and one week post injection. Histopathology showed the presence of clustered SPION microbubbles predominantly in the lungs from the first time point investigated (10 minutes). The frequency of microbubbles declined in the pulmonary vasculature and increased in pulmonary, hepatic, and splenic macrophages over time, resulting in a relative shift from the lungs to the spleen and liver. Meanwhile, macrophages showed increasing signs of cytoplasmic iron accumulation, initially in the lungs, then followed by other organs. CONCLUSION: The present investigation highlights the biological behavior of SPION microbubbles, including organ distribution over time and indications for biodegradation. The present results are essential for developing SPION microbubbles as a potential contrast agent and/or a drug delivery vehicle for specific organs. Such a vehicle will facilitate the use of multimodality imaging techniques, including ultrasound, magnetic resonance imaging, and single positron emission computed tomography, and hence improve diagnostics, therapy, and the ability to monitor the efficacy of treatment.