Porous PLGA microparticles: AI-700, an intravenously administered ultrasound contrast agent for use in echocardiography.

The production and characterization of AI-700, an intravenously administered ultrasound contrast agent under investigation for myocardial perfusion echocardiography, are described. The product consists of small, porous microparticles filled with decafluorobutane gas, and formulated as a dry powder. Small scale spray drying studies demonstrated that porous PLGA microparticles could be produced with varying porosity using ammonium bicarbonate as a volatile pore-forming agent. The porous microparticles of AI-700 were created aseptically by spray drying a water-in-oil emulsion containing poly-d,l-lactide-co-glycolide, 1,2-diarachidoyl-sn-glycero-3-phosphocholine, and ammonium bicarbonate using a two-chamber spray dryer. The porous microparticles were further formulated into a dry powder drug product (AI-700) containing decafluorobutane gas and excipients. The dry powder was reconstituted with sterile water prior to evaluation. Microscopy demonstrated that the microparticles were sphere-shaped and internally porous. The microparticles were appropriately sized for intravenous administration, having an average diameter of 2.3 mum. Zeta-potential analysis demonstrated that the microparticles would be expected to be stable post-reconstitution. The microparticles retained encapsulated gas post-reconstitution, had high acoustic potency that was stable over time and were physically stable upon exposure to high-power ultrasound, as used clinically. AI-700 has the characteristics desirable for an intravenously administered ultrasound contrast agent for myocardial perfusion echocardiography.     

触发式能量多普勒谐波成像评价犬急性心肌缺血的实验研究

目的 探讨自制的白蛋白氟碳声学造影剂对急性心肌缺血的研究价值。方法 套扎犬冠状动脉左前降支，建立急性心肌缺血模型。在套孔前后分别静脉滴注声学造影剂进行触发式能量多普勒谐波成像，评价心肌灌注结果。随后取出心脏，用0.5%伊文思蓝染色作病理对照。结果 套扎冠状动脉前，整个心肌呈环状均匀的红黄色能量显像；套扎后，前壁及部分前间隔心肌无谐波能量显像。心肌灌注缺损面积与病理染色结果差异无显著性意义。结论 静脉滴注自制的白蛋白氟碳声学造影剂通过触发式能量多普勒谐波成像能使心肌灌注显影，可用来评价急性心肌缺血。     

Physicochemical characteristics of Sonazoid, a new contrast agent for ultrasound imaging

The objective of the current work is to describe the physicochemical characteristics of Sonazoid™, a new ultrasound contrast agent for detection and characterisation of focal liver lesions. It has been demonstrated that Sonazoid™ powder for injection consists of microspheres of perfluorobutane (PFB) stabilised by a monomolecular membrane of hydrogenated egg phosphatidyl serine, embedded in an amorphous sucrose structure. Upon reconstitution with sterile water, stabilised microspheres of PFB are released in a predefined amount and size into a low viscosity, isotonic sucrose solution with a neutral pH. Sonazoid™ reconstituted product contains approximately 8 μl microspheres/ml with volume median diameter of approximately 2.6 μm. The product contains approximately 1.2 billion microspheres/ml of which less than 0.1% are larger than 7 μm. The acoustic properties of Sonazoid™ such as attenuation efficacy, fundamental and second harmonic backscatter efficacy are all well correlated to the microsphere volume concentration. The stability of Sonazoid™ after reconstitution is good, with no significant changes in physicochemical properties 2 h after reconstitution. Pressure stress is well tolerated by both concentrated and diluted Sonazoid™ with no permanent effects of pressures up to 300 mm Hg. The level and consistency of the investigated physicochemical properties demonstrate that Sonazoid™ should be well suited as a contrast agent for medical imaging with ultrasound. (E-mail: per.sontum@ge.com)     

靶向超声微泡造影剂对犬缺血再灌注心肌的延迟显像研究

目的用自制的靶向超声微泡造影剂,实现无创性地评价犬心肌缺血再灌注(I-R)的范围及严重程度.方法将本研究所自行研制的表面活性剂类超声造影剂--"表活显"表面,结合上磷脂酰丝氨酸(PS),制备成具有靶向性的造影剂(MB-PS),用MB-PS对犬I-R模型在实时心脏超声造影条件下进行延迟心肌显像,实验结束后,心肌经0.5%伊文思蓝(Evens)和1%氯化三苯四氮唑(TTC)染色,确定缺血及坏死心肌范围,并与延迟心脏超声造影显像结果比较其一致性.结果细胞流式术(FC)证明了PS结合在造影剂微泡的表面,延迟心肌显像表明缺血再灌注区的造影剂回声较正常区的回声明显增强,与病理染色结果基本一致.结论缺血损伤再灌注后,心肌缺血部位的造影剂回声较其余部位正常心肌的造影剂回声明显增强,正是因为 MB-PS聚集并停留在缺血-再灌注区,才使得超声可以发现微泡的回声,从而得以无创性地评价炎症发生的部位及其严重程度.     

Harmonic imaging of the brain parenchyma using a perfluorobutane-containing ultrasound contrast agent

We evaluated the signal-enhancing effect of the novel perfluorobutane-based ultrasound contrast agent BR 14 (Bracco Research, Switzerland) in grey-scale harmonic imaging of the brain parenchyma. Six sedated male beagle dogs were investigated with transcranial grey-scale harmonic imaging (SONOS 5500, 1.8/3.6 MHz). After bolus injection of two different doses of BR 14, acoustic densitometry was performed to quantify changes in regional contrast intensity. In the dogs' brain parenchyma, the mean relative peak increase in acoustic intensity was +61% after administration of 0.05 ml/kg BW of BR 14 and +24% after 0.2 ml/kg BW. In the masticatory muscle, application of the higher dose resulted in a stronger increase in contrast intensity compared to the lower dose. Evaluation of the contralateral base of the skull showed a dose-dependent decrease in acoustic intensity. Bolus injection of BR 14 produces an increase in acoustic intensity, which can be used for the visualization of contrast agent in the brain parenchyma. Using high dosages, a strong signal-enhancing effect in the regions near the ultrasound probe leads to a consecutive attenuation of signals from structures being located beyond ("shadowing-effect"). This is the explanation for the paradoxical result that the higher dose leads to a lower peak signal increase in the brain parenchyma.     

Potential of gold-bound microtubules as a new ultrasound contrast agent

Contrast agents based on gas-filled microspheres share the problem of time limited opacification due to low stability of microbubbles. The aim of this study was to test if gold-bound microtubules provide backscattering that allows microtubules to be potentially useful as an ultrasound (US) contrast agent. Gold colloids were immobilized on protein microtubule walls. Latex balloons were filled with gold-bound microtubules or conventional left heart contrast agent and were ultrasonographically imaged in fundamental and harmonic modes. Feasibility of anti-beta-tubulin antibody conjugation to gold-bound microtubules was confirmed using immune fluorescence analysis. Gold particles were successfully bound to microtubules. Contrast intensities in latex balloons filled with gold-bound microtubules (141 +/- 35) were comparable to those with Levovist (180 +/- 35) and did not decrease significantly during continuous US imaging for 20 min (135 +/- 34 vs. Levovist 5.0 +/- 2.0). Anti-beta-tubulin antibodies were successfully conjugated to gold-bound microtubules. Gold-bound microtubules provide a persistent contrast effect, suggesting their use as an ultrasonic contrast agent with the feasibility of antibody conjugation.     

Magnetic resonance imaging of osteosarcoma using a bis(alendronate)-based bone-targeted contrast agent

Magnetic resonance (MR) is currently used for diagnosis of osteosarcoma but not well even though contrast agents are administered. Here, we report a novel bone-targeted MR imaging contrast agent, Gd₂-diethylenetriaminepentaacetate-bis(alendronate) (Gd₂-DTPA-BA) for the diagnosis of osteosarcoma. It is the conjugate of a bone cell-seeking molecule (i.e., alendronate) and an MR imaging contrast agent (i.e., Gd-DTPA). Its physicochemical parameters were measured, including pK_a, complex constant, and T₁ relaxivity. Its bone cell-seeking ability was evaluated by measuring its adsorption on hydroxyapatite. Hemolysis was investigated. MR imaging and biodistribution of Gd₂-DTPA-BA and Gd-DTPA were studied on healthy and osteosarcoma-bearing nude mice. Gd₂-DTPA-BA showed high adsorption on hydroxyapatite, the high MR relaxivity (r₁) of 7.613 mM⁻¹ s⁻¹ (2.6 folds of Gd-DTPA), and no hemolysis. The MR contrast effect of Gd₂-DTPA-BA was much higher than that of Gd-DTPA after intravenous injection to the mice. More importantly, the MR imaging of osteosarcoma was significantly improved by Gd₂-DTPA-BA. The signal intensity of Gd₂-DTPA-BA reached 120.3% at 50 min, equal to three folds of Gd-DTPA. The bone targeting index (bone/blood) of Gd₂-DTPA-BA in the osteosarcoma-bearing mice was very high to 130 at 180 min. Furthermore, the contrast enhancement could also be found in the lung due to metastasis of osteosarcoma. Gd₂-DTPA-BA plays a promising role in the diagnoses of osteosacomas, including the primary bone tumors and metastases.     

Evaluation of a new three-dimensional magnetic imaging system for use during colonoscopy

BACKGROUND AND STUDY AIMS: A prototype magnetic imaging system (Scope Guide, Olympus Optical Co., Ltd.) provides a new facility for continuous viewing on a monitor of the position of the colonoscope during examination, without exposing patients or medical staff to radiation. The aim of this prospective study was to compare this magnetic imaging system with routine colonoscopy, including fluoroscopy. The study parameters were the detection of loops, the location of the endoscope tip at defined positions, the insertion time, and the premedication rate. MATERIALS AND METHODS: In the first part of the study, 133 consecutive patients were examined - 64 using an integrated three-dimensional colonoscope and 69 with the three-dimensional probe inserted into the biopsy channel of a routine video colonoscope. Fluoroscopy was used in all investigations for comparison at defined anatomical points and loops, and pathological findings and defined anatomic structures were documented using a laser printer both for three-dimensional colonoscopy and fluoroscopy. In the second part of the study, 25 further patients underwent colonoscopy with a modified prototype, now exclusively using the integrated three-dimensional colonoscope. RESULTS: The total time for insertion and the premedication rate did not differ from those of routine colonoscopies with fluoroscopy available. Precise detection of loops was observed in the first study in 79 - 100 % of cases in comparison with fluoroscopy. Precise localization of the endoscopic tip improved from 30 % in the first part of the study to 80 % in part 2. CONCLUSION: Using magnetic three-dimensional imaging systems, the position of the colonoscope, the detection and observation of loops during straightening, and localization of pathological findings can be accurately achieved. Modification of the prototype led to satisfactory improvement in all parameters tested.     

Parenchymal enhancement and tumor visualization using a new sonographic contrast agent.

The purpose of this study was to assess the ability of a sonographic contrast agent to increase parenchymal echogenicity and improve tumor visibility. The agent is an emulsion that changes at body temperature from nonechogenic submicrometer liquid droplets to echogenic 1 to 5 microns microbubbles, capable of transversing the pulmonary and capillary circulations. Peripheral venous injections (dosages of 0.05 to 0.8 ml/kg) were administered to five woodchucks (three with multiple hepatomas), 12 rabbits (with renal VX-2 tumors), and four dogs. Ultrasonograms were acquired from kidney, liver, tumors (including tumor vessels) and normal vessels. Uptake and washout curves were generated via videodensitometry. Finally, Doppler shifts from a cuff transducer around the celiac trunk were analyzed to provide an in vivo dose-response curve. Vascular enhancement, including hepatoma and VX-2 tumor vessels, was seen for 2 to 3 min. Maximum enhancement was 18.7 dB for a 0.6 ml/kg dose. Enhancement of normal liver and kidney parenchyma was observed in all three species for up to 20 min. Small hepatomas became more echogenic centrally, but larger tumors showed no increase in central echogenicity. In conclusion, improved tumor visibility and parenchymal enhancement was demonstrated in animals.     

Evaluation of endometrial receptivity during in-vitro fertilization using three-dimensional power Doppler ultrasound.

OBJECTIVE: To compare sonographic endometrial characteristics in in-vitro fertilization (IVF) cycles between women who conceive and those who do not. METHODS: Thirty-five women undergoing IVF treatment participated in the study. Using three-dimensional (3D) power Doppler ultrasound, we assessed endometrial patterns, volume and vascularization, after follicle stimulating hormone (FSH) stimulation but before human chorionic gonadotropin (hCG) administration (referred to hereafter as 'after FSH stimulation') and again on the day of oocyte retrieval. RESULTS: The pregnancy rate was 37% (13/35). After FSH stimulation, 29 of the 35 women had a triple-line endometrial pattern, compared with five out of 35 on the day of oocyte retrieval. In those who had a triple-line pattern after FSH stimulation the pregnancy rate was 44.8% (13/29) and it was 0% (0/6) in those with a homogeneous pattern (chi-square test, P = 0.039). If a triple-line pattern was present on the day of oocyte retrieval the pregnancy rate was 80.0% (4/5), whereas if the pattern was homogeneous the pregnancy rate was 30.0% (9/30) (P = 0.032). There were no differences between those who conceived and those who did not in endometrial thickness, volume or vascularization on either day examined. Endometrial volume decreased significantly after hCG injection in women who conceived, but not in those who did not conceive. In both groups endometrial and subendometrial vascularization decreased after hCG injection, while the endometrial thickness remained unchanged. CONCLUSIONS: The existence of a homogeneous endometrial pattern after FSH stimulation seems to be a prognostic sign of an adverse outcome in IVF, while a triple-line pattern after FSH stimulation and a decrease in endometrial volume appear to be associated with conception.     

Reproducibility of stress echocardiography using intravenous injection of ultrasound contrast agent (by 963)

Despite the widespread use of stress echocardiography, its reproducibility is still limited by high interobserver variability. Therefore, the purpose of the present study was to improve the reproducibility of a stress (exercise) echocardiography using a new transpulmonary ultrasound agent (BY 963). Stress echocardiography was performed in 12 healthy volunteers with suboptimal endocardial border delineation during exercise echocardiography. A special 45° lateral tilted bike stress echocardiography table was used for exercise testing. Echocardiographic images were recorded on-line at rest and during exercise on a video tape and additionally digitized on-line on a stress echo computer. End-diastolic (EDVml), end-systolic (ESVml) volume and ejection fraction (EF%) were estimated in the 4-chamber view. The measurements were performed before and after injection of 2.5 ml and 5 ml BY963 at rest and in maximal exercise. A new contrast agent (BY 963) leads to a sufficient contrast effect for the left ventricular cavity after intravenous administration and permits a good delineation of left the endocardial border. The interobserver variability was determined using blinded investigation by two observers. The correlation of EDV and ESV determination at rest was r = 0.68/0.33, after 2.5 ml BY 963 r = 0.97/0.93 and after 5 ml BY 963 r = 0.90/0.93. The correlation for EDV and ESV during exercise was r = 0.52/0.33, after 2.5 ml BY 963 r = 0.88/0.80 and after 5 ml BY 963 r = 0.95/0.92. At rest mean EF without contrast was 61 ± 6%/67 ± 7% (r = 0. 130), after 2.5 ml BY 963 i.v. 69 ± 8%/72 ± 7% (r = 0.82) and after 5 ml BY 963 i.v. 73 ± 8%/73 ± 8% (r = 0.98%) respectively. In exercise, mean EF without contrast was 68 ± 8%/70 ± 6 (r = 0.013), after 2.5 ml BY 963 83 ± 6%/81 ± 5 and after 5 ml 83 ± 4%/82 ± 3 (r = 0.86). Summary: The estimation of the end-systolic volume in exercise will be improved significantly and the estimated EF values will be higher compared to EF values obtained without contrast application. Transpulmonary contrast echocardiography for analysis of left ventricular volumes and ejection fraction can be routinely used in stress echocardiography. Intravenous administration of BY 963 improves the reproducibility of quantitative analysis of left ventricular function in healthy volunteers. Further studies in patients with cardiac diseases are required to corroborate this observation.     

自由解剖切面联合容积对比成像测量胎儿小脑蚓部

目的评价采用三维超声自由解剖切面结合容积对比成像(VCI)技术观察胎儿小脑蚓部的效果,与VCI-C平面技术进行比较,获取胎儿各孕周小脑蚓部发育的正常参考值。方法对196胎正常胎儿,应用三维超声自由解剖切面结合VCI技术观察胎儿小脑蚓部,测量胎儿小脑蚓部上下径、前后径和面积,分析其与孕周的关系,并与Vials等的研究结果进行比较。结果三维超声自由解剖切面结合VCI方法的测量重复性好,所测得胎儿小脑蚓部测量值均与孕周呈正相关(P<0.01),与Vials等所得测量值相近。结论三维超声自由解剖切面结合VCI技术可准确测量胎儿小脑蚓部。     

一种载磁微泡的超声/磁共振成像研究

目的制备一种超声/MRI双模态载磁微泡,并通过超声成像和MRI成像探讨载磁量对成像效果的影响。方法利用声振法将超顺磁纳米颗粒(SPIO)装载在微泡包膜上,制备超声/MRI双模态造影剂,并根据SPIO的浓度将样品分成Ⅰ～Ⅳ组(分别为0、19.6μg/ml、114.7μg/ml、292.0μg/ml),通过透射电子显微镜和紫外可见分光光度仪测量微泡基本特征和磁浓度,应用体外超声和体外MRI检测成像效果。结果透射电镜技术证实SPIO粒子成功附着,有较好的分散性,粒径呈正态分布,中心粒径约12 nm。体外超声成像显示除气水的声像图表现为无光团,未检测到回声信号;Ⅰ～Ⅳ组随着微泡浓度的增加,回声显著增强,超声图像表现为光团强度增加。Ⅰ～Ⅳ组微泡的组织对比度分别为(5.89±0.70)dB、(8.97±0.80)dB、(11.04±0.72)dB及(12.84±0.56)dB。体外MRI成像显示,T1模式下,随着微泡中磁性粒子浓度增加,其亮度增加;而在T2模式下,随着微泡中磁性粒子浓度增加,T2弛豫时间减小,亮度变弱;双模态微泡溶液和SPIO纳米粒子水溶液的纵向弛豫率分别为7.23 s~(-1)·mM~(-1)和5.43 s~(-1)·mM~(-1),横向弛豫率分别为193.62 s~(-1)·mM~(-1)和101.55 s~(-1)·mM~(-1)。结论载磁造影剂可以增强超声/MRI成像,可为优化多模态微泡造影剂的制备提供实验指导。     

Magnetic resonance imaging of tumor-associated-macrophages (TAMs) with a nanoparticle contrast agent

In the tumor micro-environment, tumor associated macrophages (TAMs) represent a predominant component of the total tumor mass, and TAMs play a complex and diverse role in cancer pathogenesis with potential for either tumor suppressive, or tumor promoting biology. Thus, understanding macrophage localization and function are essential for cancer diagnosis and treatment. Typically, tissue biopsy is used to evaluate the density and polarization of TAMs, but provides a limited “snapshot” in time of a dynamic and potentially heterogeneous tumor immune microenvironment. Imaging has the potential for three-dimensional mapping; however, there is a paucity of macrophage-targeted contrast agents to specifically detect TAM subtypes. We have previously found that sulfated-dextran coated iron oxide nanoparticles (SDIO) can target macrophage scavenger receptor A (SR-A, also known as CD204). Since CD204 (SR-A) is considered a biomarker for the M2 macrophage polarization, these SDIO might provide M2-specific imaging probes for MRI. In this work, we investigate whether SDIO can label M2-polarized cells in vitro. We evaluate the effect of degree of sulfation on uptake by primary cultured bone marrow derived macrophages (BMDM) and found that a higher degree of sulfation led to higher uptake, but there were no differences across the subtypes. Further analysis of the BMDM showed similar SR-A expression across stimulation conditions, suggesting that this classic model for macrophage subtypes may not be ideal for definitive M2 subtype marker expression, especially SR-A. We further examine the localization of SDIO in TAMs in vivo, in the mammary fat pad mouse model of breast cancer. We demonstrate that uptake by TAMs expressing SR-A scales with degree of sulfation, consistent with the in vitro studies. The TAMs demonstrate M2-like function and secrete Arg-1 but not iNOS. Uptake by these M2-like TAMs is validated by immunohistochemistry. SDIO show promise as a valuable addition to the toolkit of imaging probes targeted to different biomarkers for TAMs.

SDIO nanoparticles are localized to tumor associated macrophages (TAMs) in the mouse mammary gland breast cancer model.     

Transcranial ultrasound angiography (T USA): a new approach for contrast specific imaging of intracranial arteries

The goal was to develop an ultrasound contrast agent-specific imaging mode that offers an angiography-like view of the intracranial arteries and enables lower mechanical index MI settings compared to conventional transcranial duplex sonography. We studied 12 patients with transcranial ultrasound angiography (t USA) via the temporal bone window after an IV bolus injection of a perfluorocarbon-based microbubble contrast agent (Imagent). The aim was to display the intracranial vessel segments of the middle cerebral artery (M1, M2 and M3), the anterior cerebral artery (A1 and A2), the posterior cerebral artery (P1, P2 and P3) and the internal carotid artery (C1/2 and C3/4). t USA is a B-mode phase inversion imaging technique that uses wideband harmonic signals for image generation. We demonstrate, in this report, that t USA provides detailed anatomical display at native B-mode spatial resolution with fewer artifacts, yielding improved delineation of intracranial vessels that are in the 1- to 2-mm range.     

Ferrite particles: a new magnetic resonance imaging contrast agent. Lack of acute or chronic hepatotoxicity after intravenous administration

Intravenous administration of ferrite particles may be useful as contrast agents for magnetic resonance (MR) imaging of the liver. We studied several sensitive biochemical parameters of hepatocellular function and toxicity in rats after intravenous ferrite injections to determine whether there was any evidence of iron-induced hepatotoxicity. Light microscopy and iron determinations were performed on the liver, spleen, lung, and kidney. Forty-eight hours after a massive (250 mg iron per kilogram) ferrite injection, liver, spleen, and lung nonheme iron concentrations were markedly increased. Microscopy showed this iron to be entirely in reticuloendothelial cells. Despite the large increase in hepatic iron concentration, we found no evidence of hepatic mitochondrial or microsomal lipid peroxidation or organelle dysfunction, sensitive biochemical indicators of iron-induced hepatocellular injury. At 10 to 11 weeks after administration of ferrite in smaller doses (30 mg iron per kilogram), results of all biochemical and morphologic studies were normal. Furthermore, quantitative iron determinations and microscopic studies suggest that ferrite particles may be partially degraded and that iron is cleared from the liver during a 3-month period. Because ferrite particles are taken up by reticuloendothelial cells, hepatocellular function is not impaired and iron-induced hepatocellular injury does not occur.     

Evaluation of contrast enhancement patterns in pancreatic tumors by coded harmonic sonographic imaging with a microbubble contrast agent.

OBJECTIVE: The purpose of the study was to assess patterns of primary pancreatic lesions by contrast-enhanced sonography for differentiating ductal carcinomas from other pancreatic tumors. METHODS: One hundred six consecutive patients with pancreatic masses, consisting of 83 ductal carcinomas, 7 endocrine carcinomas, 5 intraductal papillary mucinous tumors, 3 cases of autoimmune-related pancreatitis, 3 solid pseudopapillary tumors, 2 cases of chronic pancreatitis, 1 serous cystadenoma, 1 osteoclastoid giant cell tumor, and 1 follicular lymphoma, were examined by contrast-enhanced sonography with coded harmonic imaging in a phase inversion harmonic technique. The contrast enhancement patterns were assessed, and specimens removed during pancreatectomy were subjected to pathologic examination. RESULTS: Internal tumoral vascularity was detected in 47 (56.6%) of the 83 ductal carcinomas. Vascular image spreading and homogeneous staining throughout the tumors were observed in all endocrine carcinomas. Two of the 5 intraductal papillary mucinous tumors were positive for enhancement effects. Enhancement effects were observed in all 3 cases of autoimmune-related pancreatitis, but the degree varied. There was a significant correlation between the intensity of enhancement effects and the ratio of patent vessels in the tumors (P < .05). CONCLUSIONS: Vascularity was detected by contrast-enhanced sonography in only about half of the ductal carcinomas, confirming the difficulty in distinguishing those tumors from other pancreatic tumors. There was a correlation between the patency of the vessels in the tumors and their vascularity.