Fluorous-phase iron oxide nanoparticles as enhancers of acoustic droplet vaporization of perfluorocarbons with supra-physiologic boiling point
JOURNAL OF CONTROLLED RELEASE
2019; 302: 54?62
Contrast-enhanced ultrasound of the liver: technical and lexicon recommendations from the ACR CEUS LI-RADS working group
2018; 43 (4): 861?79
Perfluorocarbon emulsion nanodroplets containing iron oxide nanoparticles (IONPs) within their inner perfluorohexane (PFH) core were prepared to investigate potential use as an acoustically activatable ultrasound contrast agent, with the hypothesis that incorporation of IONPs into the fluorous phase of a liquid perfluorocarbon emulsion would potentiate acoustic vaporization. IONPs with an oleic acid (OA) hydrophobic coating were synthesized through chemical co-precipitation. To suspend IONP in PFH, OA was exchanged with perfluorononanoic acid (PFNA) via ligand exchange to yield fluorophilic PFNA-coated IONPs (PFNA-IONPs). Suspensions with various amounts of PFNA-IONPs (0-15% w/v) in PFH were emulsified in saline by sonication, using 5% (w/v) egg yolk phospholipid as an emulsifier. PFNA-IONPs were characterized with transmission electron microscopy (TEM), transmission electron cryomicroscopy (cryoTEM), and thermogravimetric analysis (TGA) with Fourier transform infrared spectroscopy (FTIR). IONP were between 5 and 10?nm in diameter as measured by electron microscopy, and hydrodynamic size of the PFH nanodroplets were 150 to 230?nm as measured by dynamic light scattering (DLS). Acoustic droplet vaporization of PFH nanodroplets (PFH-NDs) was induced using conversion pulses (100?cycle at 1.1?MHz and 50% duty cycle) provided by a focused ultrasound transducer, and formed microbubbles were imaged using a clinical ultrasound scanner. The acoustic pressure threshold needed for PFH-NDs vaporization decreased with increasing temperature and IONP content. PFH-NDs containing 5% w/v IONP converted to microbubbles at 42?°C at 2.18 MI, which is just above the exposure limits of 1.9 MI allowed by the FDA for clinical ultrasound scanners, whereas 10 and 15% emulsion vaporized at 1.87 and 1.24 MI, respectively. Furthermore, 5% IONP-loaded PFH-NDs injected intravenously into melanoma-bearing mice at a dose of 120?mg PFH/kg, converted into detectable microbubbles in vivo 5?h, but not shortly after injection, indicating that this technique detects NDs accumulated in tumors.
View details for DOI 10.1016/j.jconrel.2019.03.013
View details for Web of Science ID 000469421500006
View details for PubMedID 30928487
Contrast-enhanced ultrasound (CEUS) liver imaging reporting and data system (LI-RADS) 2017 - a review of important differences compared to the CT/MRI system
CLINICAL AND MOLECULAR HEPATOLOGY
2017; 23 (4): 280?89
Contrast-enhanced ultrasound (CEUS) is a specific form of ultrasound imaging performed with intravenous administration of microbubble contrast agents. It has been extensively used for liver tumor characterization and was recently added to the American College of Radiology Liver Imaging Reporting and Data System (CEUS LI-RADS). This paper describes technical recommendations for successful liver CEUS lesion characterization, and provides imaging protocol and Lexicon of imaging findings.
View details for PubMedID 29151131
View details for PubMedCentralID PMC5886815
Thrombin-Activatable Microbubbles as Potential Ultrasound Contrast Agents for the Detection of Acute Thrombosis
ACS APPLIED MATERIALS & INTERFACES
2017; 9 (43): 37587?96
Medical imaging plays an important role in the diagnosis and management of hepatocellular carcinoma (HCC). The Liver Imaging Reporting and Data System (LI-RADS) was initially created to standardize the reporting and data collection of CT and MR imaging for patients at risk for HCC. As contrast-enhanced ultrasound (CEUS) has been widely used in clinical practice, it has recently been added to the LI-RADS. While CEUS LI-RADS shares fundamental concepts with CT/MRI LI-RADS, there are key differences between the modalities reflecting dissimilarities in the underlying methods of image acquisition and types of contrast material. This review introduces a recent update of CEUS LI-RADS and explains the key differences from CT/MRI LI-RADS.
View details for PubMedID 28911220
View details for PubMedCentralID PMC5760002
Polymer-Stabilized Perfluorobutane Nanodroplets for Ultrasound Imaging Agents
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2017; 139 (1): 15?18
Acute deep vein thrombosis (DVT) is the formation of a blood clot in the deep veins of the body that can lead to fatal pulmonary embolism. Acute DVT is difficult to distinguish from chronic DVT by ultrasound (US), the imaging modality of choice, and is therefore treated aggressively with anticoagulants, which can lead to internal bleeding. Here we demonstrate that conjugating perfluorobutane-filled (PFB-filled) microbubbles (MBs) with thrombin-sensitive activatable cell-penetrating peptides (ACPPs) could lead to the development of contrast agents that detect acute thrombosis with US imaging. Successful conjugation of ACPP to PFB-filled MBs was confirmed by fluorescence microscopy and flow cytometry. Fluorescein-labeled ACPP was used to evaluate the efficiency of thrombin-triggered cleavage by measuring the mean fluorescence intensity of ACPP-labeled MBs (ACPP-MBs) before and after incubation at 37 °C with thrombin. Lastly, control MBs and ACPP-MBs were infused through a tube containing a clot, and US contrast enhancement was measured with or without the presence of a thrombin inhibitor after washing the clot with saline. With thrombin activity, 91.7 ± 14.2% of the signal was retained after ACPP-MB infusion and washing, whereas only 16.7 ± 4% of the signal was retained when infusing ACPP-MBs in the presence of hirudin, a potent thrombin inhibitor.
View details for DOI 10.1021/acsami.7b10592
View details for Web of Science ID 000414506600010
View details for PubMedID 28994575
View details for PubMedCentralID PMC5691601
Contrast Enhanced Ultrasound (CEUS) Liver Imaging Reporting and Data System (LI-RADS®): the official version by the American College of Radiology (ACR).
Ultraschall in der Medizin
2017; 38 (1): 85-86
Evaluation of Knee Donor and Elbow Recipient Sites for Osteochondral Autologous Transplantation Surgery in Capitellar Osteochondritis Dissecans
AMERICAN JOURNAL OF SPORTS MEDICINE
2016; 44 (2): 511?20
In this paper, we describe a method for the stabilization of low-boiling point (low-bp) perfluorocarbons (PFCs) at physiological temperatures by an amphiphilic triblock copolymer which can emulsify PFCs and be cross-linked. After UV-induced thiol-ene cross-linking, the core of the PFC emulsion remains in liquid form even at temperatures exceeding their boiling points. Critically, the formulation permits vaporization at rarefactional pressures relevant for clinical ultrasound.
View details for DOI 10.1021/jacs.6b08800
View details for Web of Science ID 000392036900002
View details for PubMedID 28032757
In Vivo Transfection and Detection of Gene Expression of Stem Cells Preloaded with DNA-carrying Microbubbles
2015; 276 (2): 518?25
Osteochondral autologous transplantation surgery (OATS) has been advocated for treatment of osteochondritis dissecans (OCD) of the capitellum in adolescents. However, little information is available regarding the optimal knee harvest site to match the contour and cartilage thickness of the recipient elbow lesion.To characterize the capitellar anatomic structure in adolescents with and without OCD and to compare these measurements to normal adolescent knees to identify the optimal site for osteochondral graft harvest.Controlled laboratory study.Twenty-one patients with OCD were analyzed. Twenty-two patients with normal elbows and 25 age-, weight-, and height-matched patients with normal knees were also identified. Cartilage radii of curvatures (ROCs) in the sagittal and coronal-axial planes were measured on magnetic resonance imaging (MRI) of normal capitella and 5 sites (posterior lateral femoral condyle, medial and lateral middle trochlear ridges, and medial and lateral inferior trochlear ridges) in normal knees. Differences in ROC between the knee donor and capitellar recipient sites were calculated based on a 10-mm osteochondral plug diameter.Overall, the mean apex differences between graft and recipient sites ranged from 0.4 to 0.9 mm, and mean edge differences ranged from 0.5 to 1.4 mm in the coronal-axial dimension. Of all knee sites tested, the posterior lateral femoral condyle had average ROCs (19.1 mm sagittal; 14.1 mm axial) most like the capitellum (10.6 mm sagittal, 12.6 mm coronal-axial), resulting in minimal apex and edge differences (apex difference = -0.6 mm; coronal-axial side difference = -0.5 mm; no sagittal side difference). Of the anterior nonweightbearing sites, the inferior medial trochlear ridge (28.3 mm sagittal ROC; 13.2 mm coronal-axial ROC) demonstrated the lowest apex and side differences when compared with the capitellum (apex difference = -0.8 mm; coronal-axial side difference = -0.8 mm; no sagittal side difference). The frequently used middle lateral trochlear ridge (28.8 mm sagittal; 8.7 mm coronal-axial ROCs) had the largest side difference (apex distance = -0.8 mm; coronal-axial side difference = -1.4 mm; no sagittal side difference).In cases where a large single-plug OATS is considered, a 10-mm plug from the anterior nonweightbearing aspect of the distal femur is calculated to result in ?1 mm of articular incongruity at the recipient capitellum. The inferior medial trochlear ridge should be considered as a donor site for OATS procedures for OCD given its accessibility and favorable geometry.
View details for DOI 10.1177/0363546515620184
View details for Web of Science ID 000369983300033
View details for PubMedID 26712891
apoE3[K146N/R147W] acts as a dominant negative apoE form that prevents remnant clearance and inhibits the biogenesis of HDL
JOURNAL OF LIPID RESEARCH
2014; 55 (7): 1310?23
To determine whether (a) stem cells loaded with DNA-carrying microbubbles (MBs) can be transfected in vivo, (b) the cells remain alive to express the gene, and (c) gene expression is sufficiently robust to be detected in vivo.The study was approved by the Institutional Animal Care and Use Committee. Cationic MBs were prepared, characterized, and loaded with pLuciferase green fluorescent protein (GFP) plasmid. Loading was confirmed with SYBR Gold staining (Life Technologies, Carlsbad, Calif). C17.2 cells were loaded with the DNA-carrying MBs. Two hundred thousand cells suspended in 20 ?L phosphate-buffered saline were mixed with 200 ?L Matrigel (BD Biosciences, San Jose, Calif) and injected in both flanks of eight nude mice. One of the Matrigel (BD Biosciences) injections contained 50 000 cells pretransfected in vitro by using lipofectamine as a positive control. Nine flanks were exposed to 2.25-MHz ultrasonic pulses at 50% duty cycle for 1 minute at 1 W/cm(2) (n = 3) or 2 W/cm(2) (n = 6), and six flanks served as the negative control. Two days later, bioluminescent images were acquired in each mouse every 3 minutes for 1 hour after the intraperitoneal injection of d-luciferin (Perkin Elmer, Waltham, Mass). Differences between groups were assessed by using the nonparametric Kruskal-Wallis test with Wilcoxon rank sum tests for follow-up comparisons. Mice were then killed, plugs were explanted, and alternate sections were stained with hematoxylin-eosin or stained for GFP expression.Mean DNA-loaded MB diameter ± standard deviation was 2.87 ?m ± 1.69 with the DNA associated with the MB shell. C17.2 cells were associated with 2-4 MBs each, and more than 90% were viable. Peak background subtracted bioluminescent signal was fourfold higher when cells were exposed to 2 W/cm(2) pulses as compared with 1 W/cm(2) pulses (P = .02) and negative controls (P = .002). Histologic examination showed cells within the Matrigel (BD Biosciences) with robust GFP expression only after 2 W/cm(2) ultrasound exposure and lipofectamine transfection.Stem cells loaded with DNA-carrying MBs can be transfected in vivo with ultrasonic pulses and remain alive to demonstrate robust gene expression.
View details for DOI 10.1148/radiol.15141380
View details for Web of Science ID 000359796900021
View details for PubMedID 25811427
View details for PubMedCentralID PMC4521611
A(2b) Adenosine Receptor Regulates Hyperlipidemia and Atherosclerosis
2012; 125 (2): 354?U389
The K146N/R147W substitutions in apoE3 were described in patients with a dominant form of type III hyperlipoproteinemia. The effects of these mutations on the in vivo functions of apoE were studied by adenovirus-mediated gene transfer in different mouse models. Expression of the apoE3[K146N/R147W] mutant in apoE-deficient (apoE(-/-)) or apoA-I-deficient (apoA-I(-/-))×apoE(-/-) mice exacerbated the hypercholesterolemia and increased plasma apoE and triglyceride levels. In apoE(-/-) mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and caused the accumulation of discoidal apoE-containing HDL. The WT apoE3 cleared the cholesterol of apoE(-/-) mice without induction of hypertriglyceridemia and promoted formation of spherical HDL. A unique property of the truncated apoE3[K146N/R147W]202 mutant, compared with similarly truncated apoE forms, is that it did not correct the hypercholesterolemia. The contribution of LPL and LCAT in the induction of the dyslipidemia was studied. Treatment of apoE(-/-) mice with apoE3[K146N/R147W] and LPL corrected the hypertriglyceridemia, but did not prevent the formation of discoidal HDL. Treatment with LCAT corrected hypertriglyceridemia and generated spherical HDL. The combined data indicate that the K146N/R147W substitutions convert the full-length and the truncated apoE3[K146N/R147W] mutant into a dominant negative ligand that prevents receptor-mediated remnant clearance, exacerbates the dyslipidemia, and inhibits the biogenesis of HDL.
View details for DOI 10.1194/jlr.M048348
View details for Web of Science ID 000338017400011
View details for PubMedID 24776540
View details for PubMedCentralID PMC4076092
Biophysical Analysis of Apolipoprotein E3 Variants Linked with Development of Type III Hyperlipoproteinemia
2011; 6 (11): e27037
The cAMP-elevating A(2b) adenosine receptor (A(2b)AR) controls inflammation via its expression in bone marrow cells.Atherosclerosis induced by a high-fat diet in apolipoprotein E-deficient mice was more pronounced in the absence of the A(2b)AR. Bone marrow transplantation experiments indicated that A(2b)AR bone marrow cell signals alone were not sufficient to elicit this effect. Intriguingly, liver expression of the A(2b)AR in wild-type mice was vastly augmented by a high-fat diet, raising the possibility that this upregulation is of functional significance. A(2b)AR genetic ablation led to elevated levels of liver and plasma cholesterol and triglycerides and to fatty liver pathology typical of steatosis, assessed by enzymatic assays and analysis of liver sections. Western blotting and quantitative polymerase chain reaction revealed elevated expression of the following molecules in the liver of A(2b)AR-null mice: the transcription factor sterol regulatory element binding protein-1 (SREBP-1) and its 2 downstream targets and regulators of lipogenesis, acetyl CoA carboxylase and fatty acid synthase. Pharmacological activation or inhibition of A(2b)AR in primary hepatocytes confirmed the regulation of SREBP-1 by this receptor. A(2b)AR-mediated changes in cAMP were found to regulate levels of the transcriptionally active form of SREBP-1. Finally, adenovirally mediated restoration of the A(2b)AR in the liver of A(2b)AR-null mice reduced the lipid profile and atherosclerosis. Similarly, in vivo administration of the A(2b)AR ligand BAY 60-6853 in control mice on a high-fat diet reduced the lipid profile and atherosclerosis.This study provides the first evidence that the A(2b)AR regulates liver SREBP-1, hyperlipidemia, and atherosclerosis, suggesting that this receptor may be an effective therapeutic target.
View details for DOI 10.1161/CIRCULATIONAHA.111.057596
View details for Web of Science ID 000299321600025
View details for PubMedID 22144568
View details for PubMedCentralID PMC3265935
Domains of apoE4 required for the biogenesis of apoE-containing HDL
ANNALS OF MEDICINE
2011; 43 (4): 302?11
Apolipoprotein E (apoE) is a major protein of the lipoprotein transport system that plays important roles in lipid homeostasis and protection from atherosclerosis. ApoE is characterized by structural plasticity and thermodynamic instability and can undergo significant structural rearrangements as part of its biological function. Mutations in the 136-150 region of the N-terminal domain of apoE, reduce its low density lipoprotein (LDL) receptor binding capacity and have been linked with lipoprotein disorders, such as type III hyperlipoproteinemia (HLP) in humans. However, the LDL-receptor binding defects for these apoE variants do not correlate well with the severity of dyslipidemia, indicating that these variants may carry additional properties that contribute to their pathogenic potential.In this study we examined whether three type III HLP predisposing apoE3 variants, namely R136S, R145C and K146E affect the biophysical properties of the protein. Circular dichroism (CD) spectroscopy revealed that these mutations do not significantly alter the secondary structure of the protein. Thermal and chemical unfolding analysis revealed small thermodynamic alterations in each variant compared to wild-type apoE3, as well as effects in the reversibility of the unfolding transition. All variants were able to remodel multillamelar 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles, but R136S and R145C had reduced kinetics. Dynamic light scattering analysis indicated that the variant R136S exists in a higher-order oligomerization state in solution. Finally, 1-anilinonaphthalene-8-sulfonic acid (ANS) binding suggested that the variant R145C exposes a larger amount of hydrophobic surface to the solvent.Overall, our findings suggest that single amino acid changes in the functionally important region 136-150 of apoE3 can affect the molecule's stability and conformation in solution and may underlie functional consequences. However, the magnitude and the non-concerted nature of these changes, make it unlikely that they constitute a distinct unifying mechanism leading to type III HLP pathogenesis.
View details for DOI 10.1371/journal.pone.0027037
View details for Web of Science ID 000297150900046
View details for PubMedID 22069485
View details for PubMedCentralID PMC3206067
Molecular etiology of a dominant form of type III hyperlipoproteinemia caused by R142C substitution in apoE4
JOURNAL OF LIPID RESEARCH
2011; 52 (1): 45?56
INTRODUCTION. We have studied the functions of truncated apoE4 forms in vitro and in vivo in order to identify the domains of apoE4 required for the biogenesis of apoE-containing high-density lipoprotein (HDL). RESULTS. We have found that apoE4-185, -202, -229, or -259 could promote ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux in vitro, although less efficiently than Full-length apoE4, and had diminished capacity to activate lecithin cholesterol acyltransferase (LCAT). Formation of HDL in vivo was assessed by various methods following gene transfer in apolipoprotein A-I(-/-) × apoE(-/-) mice. Fast protein liquid chromatography of plasma showed that the truncated apoE forms, except apoE4-185, generated an apoE-containing HDL peak. Two-dimensional gel electrophoresis of plasma and electron microscopy showed that truncated apoE forms generated distinct HDL subpopulations and formed discoidal HDL particles which could be converted to spherical by co-administration of truncated apoE4-202 and LCAT. CONCLUSION. Overall, the in-vivo and in-vitro data are consistent and indicate that apoE4-185 is the shortest truncated form that supports formation of discoidal apoE4-containing HDL particles.
View details for DOI 10.3109/07853890.2010.549143
View details for Web of Science ID 000290935700006
View details for PubMedID 21604997
View details for PubMedCentralID PMC3110089
We have used adenovirus-mediated gene transfer in apolipoprotein (apo)E(-/-) mice to elucidate the molecular etiology of a dominant form of type III hyperlipoproteinemia (HLP) caused by the R142C substitution in apoE4. It was found that low doses of adenovirus expressing apoE4 cleared cholesterol, whereas comparable doses of apoE4[R142C] greatly increased plasma cholesterol, triglyceride, and apoE levels, caused accumulation of apoE in VLDL/IDL/LDL region, and promoted the formation of discoidal HDL. Co-expression of apoE4[R142C] with lecithin cholesterol acyltransferase (LCAT) or lipoprotein lipase (LPL) in apoE(-/-) mice partially corrected the apoE4[R142C]-induced dyslipidemia. High doses of C-terminally truncated apoE4[R142C]-202 partially cleared cholesterol in apoE(-/-) mice and promoted formation of discoidal HDL. The findings establish that apoE4[R142C] causes accumulation of apoE in VLDL/IDL/LDL region and affects in vivo the activity of LCAT and LPL, the maturation of HDL, and the clearance of triglyceride-rich lipoproteins. The prevention of apoE4[R142C]-induced dyslipidemia by deletion of the 203-299 residues suggests that, in the full-length protein, the R142C substitution may have altered the conformation of apoE bound to VLDL/IDL/LDL in ways that prevent triglyceride hydrolysis, cholesterol esterification, and receptor-mediated clearance in vivo.
View details for DOI 10.1194/jlr.M008409
View details for Web of Science ID 000285185400005
View details for PubMedID 20861163
View details for PubMedCentralID PMC2999928