Improved pH measurements with a single PARACEST MRI contrast agent

The measurement of extracellular pH has potential utility for assessing the therapeutic effects of pH‐dependent and pH‐altering therapies. A PARAmagnetic chemical exchange saturation transfer (PARACEST) MRI contrast agent, Yb–DO3A–oAA, has two CEST effects that are dependent on pH. A ratio derived from these CEST effects was linearly correlated with pH throughout the physiological pH range. The pH can be measured with a precision of 0.21 pH units and an accuracy of 0.09 pH units. The pH measurement is independent of concentration and T₁ relaxation times, but is dependent on temperature. Although MR coalescence affects the CEST measurements, especially at high pH, the ratiometric analysis of the CEST effects can account for incomplete saturation of the agent's amide and amine that results from MR coalescence. Provided that an empirical calibration is determined with saturation conditions, magnetic field strength and temperature that can be used for subsequent studies, these results demonstrate that this single PARACEST MRI contrast agent can accurately measure pH. Copyright © 2012 John Wiley & Sons, Ltd.     

碘造影剂脂质体的研究进展

碘造影剂广泛应用于临床影像诊断,研究将其包裹于脂质体中具有提高组织器官造影的靶向性、延长强化时间等作用,国外对此研究已有20多年,有些已经进入临床试验,但至今未有上市产品.国内在这方面研究较少,有也仅限于实验室研究.本文主要就国内外碘造影剂脂质体的制备方法以及相关的药效学研究作一综述.     

Effect of ultrasound parameters on the release of liposomal calcein

The ultrasound exposure parameters that maximize drug release from dierucoyl-phosphatidylcholine (DEPC)-based liposomes were studied using two transducers operating at 300 kHz and 1 MHz. Fluorescent calcein was used as a model drug, and the release from liposomes in solution was measured using a spectrophotometer. The release of calcein was more efficient at 300 kHz than at 1 MHz, with thresholds of peak negative pressures of 0.9 MPa and 1.9 MPa, respectively. Above this threshold, the release increased with increasing peak negative pressure, mechanical index (MI), and duty cycle. The amount of drug released followed first-order kinetics and increased with exposure time to a maximal release. To increase the release further, the MI had to be increased. The results demonstrate that the MI and the overall exposure time are the major parameters that determine the drug's release. The drug's release is probably due to mechanical (cavitation) rather than thermal effects, and that was also confirmed by the detection of hydroxide radicals.     

Novel reduction-sensitive micelles for triggered intracellular drug release

Novel reduction-sensitive micelles based on poly(ethylene oxide)-b-poly(N-methacryloyl-N′-(t-butyloxycarbonyl)cystamine) (PEO-b-PMABC) diblock copolymers were developed and applied for triggered intracellular drug release. PEO-b-PMABC block copolymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of MABC with dithioester-capped PEO as macroRAFT agent. Gel permeation chromatography (GPC) and ¹H NMR analysis showed that the copolymers have controlled compositions and molecular weights, indicating the living nature of polymerization. These copolymers were self-assembled into micelles. The physicochemical characteristics and reduction-sensitivity of the resultant micelles were investigated by fluorescence measurement, transmission electron microscopy (TEM), and dynamic light scattering (DLS). The results showed that PEO-b-PMABC micelles are stable at normal physiologic condition but readily cleaved into free copolymers under reducing environment. In vitro release of doxorubicin (DOX) and cell experiments showed that the drug-loaded PEO-b-PMABC micelles accomplished much faster drug release under reducing condition and higher anticancer efficacy as compared to the control without reduction-sensitivity, indicating great potential of PEO-b-PMABC micelles for efficient intracellular drug delivery.     

A method for accurate pH mapping with chemical exchange saturation transfer (CEST) MRI

Chemical exchange saturation transfer (CEST) MRI holds enormous promise for imaging pH. Whereas the routine CEST‐weighted MRI contrast is complex and susceptible to confounding factors such as labile proton ratio, chemical shift, bulk water relaxation and RF saturation, ratiometric CEST imaging simplifies pH determination. However, the conventional ratiometric CEST (RCEST) MRI approach is limited to CEST agents with multiple exchangeable groups. To address this limitation, RF power‐based ratiometric CEST (PRCEST) imaging has been proposed that ratios CEST effects obtained under different RF power levels. Nevertheless, due to concomitant RF saturation (spillover) effect, the recently proposed PRCEST imaging is somewhat dependent on parameters including bulk water relaxation time and chemical shift. Herein we hypothesized that RF power‐based ratiometric analysis of RF spillover effect‐corrected inverse CEST asymmetry (PRICEST) provides enhanced pH measurement. The postulation was verified numerically, and validated experimentally using an in vitro phantom. Briefly, our study showed that the difference between MRI‐determined pH (pH_MRI) and electrode‐measured pH being 0.12 ± 0.13 and 0.04 ± 0.03 for PRCEST and PRICEST imaging, respectively, and the newly proposed PRICEST imaging provides significantly more accurate pH determination than PRCEST imaging (P < 0.01, Wilcoxon signed‐rank test). Notably, the exchange rate shows dominantly base‐catalysed relationship with pH, independent of creatine concentration (P > 0.10, Analysis of Covariance). In addition, the derived labile proton ratio linearly scales with creatine concentration (P < 0.01, Pearson Regression). To summarize, PRICEST MRI provides concentration‐independent pH imaging, augmenting prior quantitative CEST methods for accurate pH mapping. Copyright © 2015 John Wiley & Sons, Ltd.     

Application of liposomes to sonoporation

A method to prepare liposomes is presented. Liposomes made in our laboratory were characterized acoustically and optically. The phase velocity and attenuation of liposomes in suspension (concentration = 10(9)/mL) were measured, ranging from 2 to 14 MHz, using ultrasound spectroscopy. Anti-rabbit IgG conjugated with Alexafluor 647 was delivered into Jurkat cells in suspension, using the liposomes, by 10 % duty cycle ultrasound tonebursts of 2.2 MHz (the in situ spatial peak-pressure amplitude = 80 W/cm2) with an efficiency of 13 %. It has been experimentally shown that liposomes may be an alternative stable agent to Optison for delivering macromolecules into cells.     

颅脑肿瘤磁共振扫描增强的价值(附72例分析)

目的：颅脑肿瘤注入Ｇｄ－ＤＴＰＡ（磁显葡胺）以区别瘤体与水肿，鉴别肿瘤良、恶性及囊壁，显示脑转移瘤和子瘤灶，达到临床诊断目的；资料和方法：７２例颅脑肿瘤，其中脑内肿瘤４８例，脑外肿瘤２４例。均用ＡＳＭ－０１５Ｐ永磁型ＭＲＩ扫描机，采用头线圈，ＳＥ序列：Ｔ１加权像ＴＲ＝５００ｍｓ，ＴＥ＝３０ｍｓ，Ｔ２加权像ＴＲ＝２０００－３０００ｍｓ，ＴＥ＝１００－１２０ｍｓ，，层厚５－１０ｍｍ，方位行横、冠、矢状位，增强扫描为：静脉缓慢注入Ｇｄ－ＤＴＰＡ（磁显葡胺）按０１－０２ｍｌ／ｋｇ体重，注药后５、１０、２５、３５ｍｉｎ作相应方位Ｔ１加权像扫描；结果：１脑内肿瘤４８例，胶质瘤３３例，明显及部分强化２５例，不强化８例，脑转移１５例均强化共３９个瘤灶（其中新发现８个）；（２）脑外肿瘤２４例，脑膜瘤１４例，垂体瘤６例，听神经瘤３例上述均强化，硬膜外表皮样囊肿１例不强化；结论：颅脑肿瘤通过静脉注入Ｇｄ－ＤＴＰＡ（磁显葡胺）可以明确瘤灶与水肿及发现Ｔ２加权像上被水肿掩盖的子灶，达到临床诊断目的。     

Dynamic changes in 1H‐MR relaxometric properties of cell‐internalized paramagnetic liposomes,as studied over a five‐day period

Molecular imaging based on MRI requires the use of amplification strategies in order to achieve sufficient sensitivity for the detection of low‐level molecular markers. Recently, we described a combination of two amplification methods: (i) the use of paramagnetic liposomes that can be prepared with a high payload of Gd³⁺‐containing lipid; and (ii) targeting to a cell‐surface receptor that can undergo multiple rounds of nanoparticle delivery in the cell, followed by recycling to the cell membrane. Liposome uptake was monitored over a period of 24 h and was found to lead to massive delivery in subcellular compartments. The present study aimed to monitor the longer‐term fate of the cell‐internalized contrast material by studying its relaxometric properties over 5 days, following an initial 24 h loading period. Circa 25% of the Gd³⁺‐content delivered to the cells via integrin‐targeted liposomes was lost in the first 24 h, which led to 65 and 77% reductions in R₁ and R₂, respectively, as compared with the original R₁ and R₂ enhancements. This implies that the remaining cell‐associated gadolinium had relatively low effective r₁ and r₂ relaxivities. It is proposed that this is due to gradual release of Gd³⁺ from the chelate in the cell, followed by sequestration in an MR silent state. Most of the gadolinium internalized by cells following incubation with non‐targeted liposomes was released in the 5‐day follow‐up period. Copyright © 2010 John Wiley & Sons, Ltd.     

Coupling of drug containing liposomes to microbubbles improves ultrasound triggered drug delivery in mice

Local extravasation and triggered drug delivery by use of ultrasound and microbubbles is a promising strategy to target drugs to their sites of action. In the past we have developed drug loaded microbubbles by coupling drug containing liposomes to the surface of microbubbles. Until now the advantages of this drug loading strategy have only been demonstrated in vitro. Therefore, in this paper, microbubbles with indocyanine green (ICG) containing liposomes at their surface or a mixture of ICG-liposomes and microbubbles was injected intravenously in mice. Immediately after injection the left hind leg was exposed to 1 MHz ultrasound and the ICG deposition was monitored 1, 4 and 7 days post-treatment by in vivo fluorescence imaging. In mice that received the ICG-liposome loaded microbubbles the local ICG deposition was, at each time point, about 2-fold higher than in mice that received ICG-liposomes mixed with microbubbles. We also showed that the perforations in the blood vessels allow the passage of ICG-liposomes up to 5 h after microbubble and ultrasound treatment. An increase in tissue temperature to 41 °C was observed in all ultrasound treated mice. However, ultrasound tissue heating was excluded to cause the local ICG deposition. We concluded that coupling of drug containing liposomes to microbubbles may increase ultrasound mediated drug delivery in vivo.     

动态增强磁共振成像对正常人肾功能的评估

目的　评估动态增强磁共振检查对正常肾脏肾功能评价的作用。方法　 15名正常志愿者 ,三天内行血清肌酐(Scr)及尿素氮 (BUN)测定及肾脏动态增强MRI检查。肾脏功能分析为在动态增强MR影像上选择感兴趣区测量各肾皮质及髓质的信号强度 ,绘出各部位平均信号强度 时间曲线 ,记录皮质、髓质的平均峰值及峰值 /达峰时间比 ,研究这些参数与Scr及BUN的相关性。结果　皮质信号峰值及髓质信号峰值与Scr呈显著线性相关 (P <0 .0 0 1,r =-0 .78,r =-0 .76)。结论　动态增强MRI作为评估正常肾脏灌注及肾小管浓缩功能的无创性影像学方法 ,可综合反映肾脏结构及功能。     

Imaging analysis for multiple paramagnetic agents using OMRI and electrophoresis

Nitroxides have been widely used as a molecular probe for analysis of various diseases models. This article describes an analytical method for separation and semi-quantification of multiple paramagnetic contrast agents with simple procedure combining electrophoresis and Overhauser enhancement magnetic resonance imaging (OMRI) imaging. We used three nitroxides, 3-carbamoyl PROXYL, 3-carboxy PROXYL, and CAT-1, which have different ionic charges in the molecule. In addition, we showed that this method could apply for in vitro measurement using biological sample. The results showed the nitroxides were successfully separated with electrophoresis depending on their charge, and their separation was visualized with OMRI after electrophoresis. Vehicle media such as whole blood did not affect the electrophoresis results and OMRI enhancement factor. Thus, the method can be used to analyze the redox status of biological samples without preprocessing. This analytical method enables in vitro measurement of biological samples to determine the redox status of specific tissue layers using paramagnetic agents, which is helpful for detailed analysis of redox-related diseases.     

A method to co-encapsulate gas and drugs in liposomes for ultrasound-controlled drug delivery

We describe a novel method for the facile production of gas-containing liposomes with simultaneous drug encapsulation. Liposomes of phospholipid and cholesterol were prepared by conventional procedures of hydrating the lipid film, sonicating, freezing and thawing. A single but critical modification of this procedure generates liposomes that contain gas (air, perfluorocarbon, argon); after sonication, the lipid is placed under pressure with the gas of interest. After equilibration, the sample is frozen. The pressure is then reduced to atmospheric and the suspension thawed. This procedure leads to entrapment of air in amounts up to 10% by volume in lipid dispersions at moderate (10 mg/mL) concentrations of lipids. The amount of gas encapsulated increases with gas pressure and lipid concentration. Using 0.32 mol/L mannitol to provide an aqueous phase with physiological osmolarity, 1, 3, 6 or 9 atm of pressure was applied to 4 mg of lipid. This led to encapsulation of 10, 15, 20 and 30 microl of gas in a total of 400 microl of liposome dispersion (10 mg lipids/mL), respectively. The mechanism for gas encapsulation presumably depends on the fact that air (predominantly nitrogen and oxygen), like most solutes, dissolves poorly in ice and is excluded from the ice that forms during freezing. The excluded air then comes out of solution as air pockets that are stabilized in some form by a lipid coating. The presence of air in these preparations sensitizes them to ultrasound (1MHz, 8 W/cm2,10 s) such that up to half of their aqueous contents (which could be a water soluble drug) can be released by short (10 s) applications of ultrasound. Both diagnostic and therapeutic applications of the method are conceivable.     

Massage-induced release of subcutaneously injected liposome-encapsulated drugs to the blood

Liposome-based, externally regulated drug delivery system is described in which liposome-encapsulated bioactive molecules can be delivered into the blood in response to simple mechanical action. Without any mechanical stimulation, subcutaneously injected 200 nm liposomes are usually trapped in the interstitial space for prolonged time. However, upon lymphotropic stimulation (such as manual massage of the injection site), the liposomes can be mobilized into the blood via lymphatic pathway. Up to 40% of the injected dose can be delivered to the blood via lymphatic pathway from the injection site at the rabbit's front paw dorsum during 5 min manual massage cycle. Using vasoconstricting hormone angiotensin II as liposome-encapsulated pharmacological marker, we demonstrated that physiological response to encapsulated drug (average blood pressure increase) can also be induced and modulated by massage. Massage itself was found to have no effect on the blood pressure. Modification of liposome surface with polyethylene glycol was found to increase blood localization of the liposome-encapsulated drug presumably due to decreasing the uptake of the drug carrier by lymph node macrophages. Pressure-dependent gaps between lymphatic capillary endothelial cells are thought to play the role of the size discrimination device allowing larger particulates into the lymphatics and, eventually, into the blood after increase of interstitial pressure caused by injection site massage.     

DOTAM‐type ligands possessing arginine pendant groups for use in PARACEST MRI

A synthetic methodology was developed for the preparation of metal‐chelating ligands that possess arginine pendant groups relying on the alkylation of 1,4,7,10‐tetraazacyclododecane (cyclen) with arginine‐containing electrophiles. Conditions for the selective trialkylation or peralkylation of cyclen are described, the outcome being dependent on the nature of the arginine‐derived electrophile and the solvent used for the reaction. Lanthanide metal complexes of the ligands prepared by the described route were evaluated for their suitability as PARACEST contrast agents for use in magnetic resonance imaging. The Dy³⁺ and Tm³⁺ complexes display CEST effects that are associated with the amide protons proximate to the metal center. These signals exhibit pH dependence in the range of 6.0–8.0 and thus may have the potential for pH measurement in physiological range. Copyright © 2012 John Wiley & Sons, Ltd.     

The pH sensitivity of –NH exchange in LnDOTA–tetraamide complexes varies with amide substituent

The amide proton exchange rates in various lanthanide(III) DOTA–tetraamide complexes were investigated by CEST as a function of variable chemical structures and charges on the amide substituents. Comparisons were made between YbDOTA–(gly)₄^? (Yb‐1), YbDOTA–(NHCH₂PO₃)₄^5? (Yb‐2) and YbDOTA–(NHCH₂PO₃Et₂)₄³⁺ (Yb‐3). The general shapes of the CEST vs pH profiles were similar for the three complexes but they showed maximum CEST intensities at different pH values, pH 8.3, 8.8 and 6.9 for Yb‐1, Yb‐2 and Yb‐3, respectively. This indicates that a more negatively charged substituent on the amide helps stabilize the partial positive charge on the amide nitrogen and consequently more base is required to catalyze proton exchange. The chemical shifts of the –NH protons in Yb‐1 and Yb‐2 were similar (?17 ppm) while the –NH proton in Yb‐3 was at ?13 ppm. This shows that the crystal field produced by the amide oxygen donor atoms in Yb‐3 is substantially weaker than that in the other two complexes. In an effort to expand the useful range of pH values that might be measured using these complexes as CEST agents, the shapes of the CEST vs pH curves were also determined for two thulium(III) complexes with much larger hyperfine shifted –NH proton resonances. The ratio of CEST from –NH exchange in Tm‐1 compared with CEST from –NH exchange in Tm‐3 was found to be linear over an extended pH range, from 6.3 to 7.4. This demonstrates a potential advantage of using mixtures of lanthanide(III) DOTA–tetraamides for mapping tissue pH by use of ratiometric CEST imaging. Copyright © 2011 John Wiley & Sons, Ltd.     

Characterization of Acoustic Properties of PVA-Shelled Ultrasound Contrast Agents: Ultrasound-Induced Fracture (Part II)

Knowledge of the magnitude of the peak negative pressure, P_thr, at which ultrasound contrast agents fracture is relevant for using these microbubbles both as devices for contrast enhancement purposes, as well as carriers of drugs to be delivered locally. In the second part of this communication, the acoustic properties of three types of microbubbles stabilized by poly (vinyl alcohol) (PVA) shells are further investigated. In particular, the dependence of P_thr on system parameters such as the number of cycles, frequency and exposure is examined. The effects of temperature, blood and, wherever data are available, of the dimension of the microbubbles on P_thr are also considered. The large shell thickness notwithstanding, the results of this investigation show that at room temperature, PVA contrast agents fracture at negative peak pressure values within the recommended safety limit. Furthermore, P_thr decreases with increasing temperature, radius of the microbubbles and number of cycles of the incident wave. Fatigue seems to be a physical mechanism playing a dominant role in the fracture process. The effect of blood on P_thr varies according to condition under which the microbubbles have been synthesized, although stiffening of the shell is observed in most cases. In conclusion, these results suggest that PVA-shelled microbubbles may offer a potentially viable system to be employed for both imaging and therapeutic purposes.     

磁共振增强扫描对颅后窝占位性病变 的诊断价值

目的 评价增强扫描对颅后窝占位性病变定性诊断及术后随访的价值。方法 回顾性分析38例颅后窝占位性病变的MRI平扫及增强表现。全部病例均经手术病理及临床证实,其中星形细胞瘤8例。髓骨细胞瘤3例。血管母细胞瘤4例。转移瘤5例,脑膜瘤9例,脑脓肿2例,表皮样囊肿3例。蛛网膜囊肿4例。结果 血管母细胞瘤呈大囊小结节型。壁结节显著均匀强化,囊壁囊液不强化;髓母细胞瘤实质强化较明显,内部囊变区较小;星形细胞瘤可呈实质性或囊实性,强化表现复杂;转移瘤及脑脓肿强化表现相似,可结合病史加以鉴别;脑膜瘤明显强化,并可显示：脑膜尾征“,表皮样囊肿和蛛网膜囊肿不强化,9例患者术后进行了11次复查,1例诊断肿瘤残留复发,8例可见不同程度的术后改变。结论 MRI增强扫描能对大多数颅后窝占位性病变进行定性诊断,对临床治疗及术后随访具有较高的指导意义。     

Characterisation of Liposome-Loaded Microbubble Populations for Subharmonic Imaging

Therapeutic microbubbles could make an important contribution to the diagnosis and treatment of cancer. Acoustic characterisation was performed on microfluidic generated microbubble populations that either were bare or had liposomes attached. Through the use of broadband attenuation techniques (3–8 MHz), the shell stiffness was measured to be 0.72 ± 0.01 and 0.78 ± 0.05 N/m and shell friction was 0.37 ± 0.05 and 0.74 ± 0.05 × 10^?6 kg/s for bare and liposome-loaded microbubbles, respectively. Acoustic scatter revealed that liposome-loaded microbubbles had a lower subharmonic threshold, occurring from a peak negative pressure of 50 kPa, compared with 200 kPa for equivalent bare microbubbles. It was found that liposome loading had a negligible effect on the destruction threshold for this microbubble type, because at a mechanical index >0.4 (570 kPa), 80% of both populations were destroyed.     

原发性肝癌的MRI征象与肝癌转移发生的相关性分析

目的　总结原发性肝癌的MRI征象 ,分析其与肝癌转移之间的相关性。方法　收集我院经病理确诊为原发性肝癌的患者 5 0 0例 ,分别行平扫、Gd DTPA及SPIO增强扫描。结果　小肝癌 65例 (13 % ) ,结节型 81例 (16.2 % ) ,块状型 3 2 5例(65 % ) ,弥漫型 2 9例 (5 .8% )。多发肿瘤 190例 (3 8% )。淋巴结转移率为 12 % ;静脉癌栓为 3 1.4%。结论　原发性肝癌的MRI征象中大病灶、多病灶、T1信号不均匀者发生转移的几率较大 ,而四种转移形式之一均提示其他转移的几率增加。     

超声造影与MRI诊断乳头溢液性病变的对比研究

目的探讨超声造影和MRI检查在乳头溢液性病变诊断中的影像学特征。方法对26例乳头溢液患者行超声造影和MRI检查,分析两种检查方法的图像特征,比较两种检查方法对病灶良恶性的诊断符合率。结果超声造影和MRI对病灶诊断的敏感性、特异性、阳性预测值、阴性预测值及准确性分别为81.81%、86.67%、81.81%、86.67%、84.62%以及63.64%、80.00%、70.00%、75.00%、73.08%。结论超声造影和MRI对乳头溢液性病变性质的诊断均有重要价值。