Long-term survival following a single treatment of kidney tumors with multiwalled carbon nanotubes and near-infrared radiation

Multiwalled carbon nanotubes (MWCNTs) exhibit physical properties that render them ideal candidates for application as noninvasive mediators of photothermal cancer ablation. Here, we demonstrate that use of MWCNTs to generate heat in response to near-infrared radiation (NIR) results in thermal destruction of kidney cancer in vitro and in vivo. We document the thermal effects of the therapy through magnetic resonance temperature-mapping and heat shock protein-reactive immunohistochemistry. Our results demonstrate that use of MWCNTs enables ablation of tumors with low laser powers (3 W/cm²) and very short treatment times (a single 30-sec treatment) with minimal local toxicity and no evident systemic toxicity. These treatment parameters resulted in complete ablation of tumors and a >3.5-month durable remission in 80% of mice treated with 100 μg of MWCNT. Use of MWCNTs with NIR may be effective in anticancer therapy.     

Diameter and rigidity of multiwalled carbon nanotubes are critical factors in mesothelial injury and carcinogenesis

Multiwalled carbon nanotubes (MWCNTs) have the potential for widespread applications in engineering and materials science. However, because of their needle-like shape and high durability, concerns have been raised that MWCNTs may induce asbestos-like pathogenicity. Although recent studies have demonstrated that MWCNTs induce various types of reactivities, the physicochemical features of MWCNTs that determine their cytotoxicity and carcinogenicity in mesothelial cells remain unclear. Here, we showed that the deleterious effects of nonfunctionalized MWCNTs on human mesothelial cells were associated with their diameter-dependent piercing of the cell membrane. Thin MWCNTs (diameter ～ 50 nm) with high crystallinity showed mesothelial cell membrane piercing and cytotoxicity in vitro and subsequent inflammogenicity and mesotheliomagenicity in vivo. In contrast, thick (diameter ～ 150 nm) or tangled (diameter ～ 2-20 nm) MWCNTs were less toxic, inflammogenic, and carcinogenic. Thin and thick MWCNTs similarly affected macrophages. Mesotheliomas induced by MWCNTs shared homozygous deletion of Cdkn2a/2b tumor suppressor genes, similar to mesotheliomas induced by asbestos. Thus, we propose that different degrees of direct mesothelial injury by thin and thick MWCNTs are responsible for the extent of inflammogenicity and carcinogenicity. This work suggests that control of the diameter of MWCNTs could reduce the potential hazard to human health.     

Detecting insulitis in type 1 diabetes with ultrasound phase-change contrast agents

Type 1 diabetes (T1D) results from immune infiltration and destruction of insulin-producing β cells within the pancreatic islets of Langerhans (insulitis). Early diagnosis during presymptomatic T1D would allow for therapeutic intervention prior to substantial β-cell loss at onset. There are limited methods to track the progression of insulitis and β-cell mass decline. During insulitis, the islet microvasculature increases permeability, such that submicron-sized particles can extravasate and accumulate within the islet microenvironment. Ultrasound is a widely deployable and cost-effective clinical imaging modality. However, conventional microbubble contrast agents are restricted to the vasculature. Submicron nanodroplet (ND) phase-change agents can be vaporized into micron-sized bubbles, serving as a microbubble precursor. We tested whether NDs extravasate into the immune-infiltrated islet microenvironment. We performed ultrasound contrast-imaging following ND infusion in nonobese diabetic (NOD) mice and NOD;Rag1ko controls and tracked diabetes development. We measured the biodistribution of fluorescently labeled NDs, with histological analysis of insulitis. Ultrasound contrast signal was elevated in the pancreas of 10-wk-old NOD mice following ND infusion and vaporization but was absent in both the noninfiltrated kidney of NOD mice and the pancreas of Rag1ko controls. High-contrast elevation also correlated with rapid diabetes onset. Elevated contrast was also observed as early as 4 wk, prior to mouse insulin autoantibody detection. In the pancreata of NOD mice, infiltrated islets and nearby exocrine tissue were selectively labeled with fluorescent NDs. Thus, contrast ultrasound imaging with ND phase-change agents can detect insulitis prior to diabetes onset. This will be important for monitoring disease progression, to guide and assess preventative therapeutic interventions for T1D.

Type 1 diabetes (T1D) is caused by infiltration of autoreactive immune cells into the islets of Langerhans in the pancreas (insulitis) and destruction of insulin-secreting β-cells. The subsequent loss of glucose homeostasis requires lifelong insulin therapy, with T1D subjects still at an elevated risk of chronic diabetes complications and hypoglycemia-induced coma or death. Prior to clinical onset, there exists an asymptomatic phase of many years where autoimmunity and insulitis progresses but glucose homeostasis is maintained (presymptomatic T1D) (1). However, patients with T1D are not diagnosed until presentation of hyperglycemia, where the majority of β-cell mass (>80%) has been lost. As such, this presymptomatic phase of T1D provides an ideal window for therapeutic prevention (2).There has been some limited success in trials applying immunomodulatory agents designed to preserve β-cell mass and reverse or prevent T1D (3). For example, antiCD3 has recently been applied in first-degree relatives at high risk for T1D and showed a significant delay and reduced incidence of T1D onset (4). However, prevention was not complete, and these subjects were still significantly advanced in T1D progression, showing signatures of dysglycemia. Application of therapies at T1D onset similarly show only a temporary preservation of β-cell mass (as measured by c-peptide release), with a broad heterogeneity in treatment response (4). As such, earlier therapeutic treatment is desirable.There are currently very limited means to determine whether a subject shows presymptomatic T1D. Furthermore, there are no means to assess the success or failure of therapeutic interventions beyond the onset of T1D itself. The presence of multiple islet-associated autoantibodies in circulation can determine risk of developing T1D within a multiyear time frame (5, 6). However, islet-associated autoantibodies are not pathogenic and cannot inform on successful therapeutic treatments. As such, there is a need to develop new methods to track the progression of insulitis and β-cell mass decline during presymptomatic T1D. Furthermore, at clinical onset there is a high incidence of diabetic ketoacidosis (DKA) that can have severe health consequences (7). Thus identifying “at-risk” subjects and monitoring their underlying disease progression will be important to mitigate the consequences of DKA.Several imaging approaches have been explored to detect insulitis and β-cell mass decline (8–11). During insulitis, the islet microvasculature increases permeability (11, 12). The enhanced permeability and retention (EPR) effect can therefore be used, where submicron-sized agents can accumulate in the inflamed islet microenvironment. This effect has been demonstrated using MRI contrast agents, in both mouse models of T1D and human T1D (13, 14). While promising, MRI modalities carry some limitations in terms of deployability and cost-effectiveness. Ultrasound imaging modalities can be widely deployed, are cost-effective, and provide real-time imaging. Contrast-enhanced ultrasound uses gas filled microbubbles and nonlinear signal detection. However, conventional microbubble contrast agents are restricted to the vasculature. Submicron-sized nanobubble ultrasound contrast agents have been developed that accumulate in tumors and inflamed tissue, including the immune infiltrated pancreas (15, 16). However, one potential limitation with submicron-sized bubbles is their low scattering cross-section, requiring a larger number of bubbles to be infused to generate measurable signal.Nanodroplet phase-change agents are a class of submicron-sized ultrasound contrast agents (17, 18). Nanodroplets consist of a superheated gas core and folded lipid shell that are stable at body temperature. However, within an acoustic beam these nanodroplets (NDs) can vaporize into micron-sized bubbles that provide significant ultrasound contrast. As such, these NDs can act as a circulating nanoscale microbubble “precursor” to be “activated” by an acoustic beam. Nanodroplets can be readily formed by condensing conventional microbubble agents (19), including clinically approved agents (20). Given their submicron size, NDs have been demonstrated to follow the EPR effect and accumulate in tumors and generate measurable ultrasound contrast signals (19, 21). However, whether NDs can similarly accumulate in inflamed tissues and whether this accumulation can be measured and used for disease monitoring has been lacking.In this study, we tested whether ND phase-change contrast agents specifically accumulate within inflamed islets of Langerhans in mouse models of T1D and the degree to which this accumulation depends on the level of insulitis. We further tested whether this accumulation could be measured noninvasively via ultrasound contrast imaging, as an increase in contrast signal following ND vaporization, and how this correlated with diabetes onset.     

Nanomechanical mechanism for lipid bilayer damage induced by carbon nanotubes confined in intracellular vesicles

Understanding the behavior of low-dimensional nanomaterials confined in intracellular vesicles has been limited by the resolution of bioimaging techniques and the complex nature of the problem. Recent studies report that long, stiff carbon nanotubes are more cytotoxic than flexible varieties, but the mechanistic link between stiffness and cytotoxicity is not understood. Here we combine analytical modeling, molecular dynamics simulations, and in vitro intracellular imaging methods to reveal 1D carbon nanotube behavior within intracellular vesicles. We show that stiff nanotubes beyond a critical length are compressed by lysosomal membranes causing persistent tip contact with the inner membrane leaflet, leading to lipid extraction, lysosomal permeabilization, release of cathepsin B (a lysosomal protease) into the cytoplasm, and cell death. The precise material parameters needed to activate this unique mechanical pathway of nanomaterials interaction with intracellular vesicles were identified through coupled modeling, simulation, and experimental studies on carbon nanomaterials with wide variation in size, shape, and stiffness, leading to a generalized classification diagram for 1D nanocarbons that distinguishes pathogenic from biocompatible varieties based on a nanomechanical buckling criterion. For a wide variety of other 1D material classes (metal, oxide, polymer), this generalized classification diagram shows a critical threshold in length/width space that represents a transition from biologically soft to stiff, and thus identifies the important subset of all 1D materials with the potential to induce lysosomal permeability by the nanomechanical mechanism under investigation.The interactions of low-dimensional materials with the external or plasma membrane of living cells have been the subject of prior studies due to their importance in uptake and delivery, antibacterial action, and nanomaterial safety (1–6). Following uptake, nanomaterials may also interact with internal membranes while under confinement in intracellular vesicles (7–10), but the biophysics of these geometrically constrained systems is poorly understood. Low-dimensional materials interact with biological systems in complex ways dictated by their 1D nanofibrous or 2D nanosheet geometries (7, 11–20). These interactions typically begin when materials encounter the plasma membrane and initiate phenomena that can include adhesion, membrane deformation, penetration, lipid extraction, entry, frustrated uptake, or cytotoxicity (4, 11–14, 19–21). Recent experimental data suggest that the cellular response to some 1D materials is governed by their interaction with the internal lipid-bilayer membranes of endosomes and lysosomes following nanomaterial uptake (7–10). The resulting geometry is fundamentally different in that the fibrous materials are confined within a vesicle, imposing geometric constraints and introducing mechanical forces that act bidirectionally––i.e., on both the thin fibrous structure and the inner leaflet of the soft membrane. The fundamental biophysics of this tube-in-vesicle system is virtually unexplored, yet may be critical for understanding the cellular response to nanotubes/fibers, where shape and stiffness are among the known determinants of toxicity (13, 21). The technique of coarse-grained molecular dynamics (MD), demonstrated to be effective in the study of complex biomolecular systems (22, 23), has been applied to whole lipid-bilayer patches to reveal a biophysical mechanism for carbon nanotube interaction with the plasma membranes leading to tip entry and uptake (4, 19, 20). The same technique may also provide insight relevant to internal membrane interactions, although whole vesicle MD is a significant challenge. Here we use a complement of techniques including coarse-grained MD, all-atom MD, in vitro bioimaging, and carbon nanotube length modification to reveal the behavior of vesicle-encapsulated carbon nanotubes and identify the conditions and carbon nanotube (CNT) types that lead to mechanical stress and membrane damage following cellular uptake and packaging in lysosomes (8).     

Established and emerging roles for ultrasound enhancing agents (contrast echocardiography)

The ability to opacify the left ventricle and delineate the endocardium after intravenous injection of microbubble ultrasound enhancing agents is of established value to quantify volumes and function in suboptimal unenhanced images, particularly in stress echocardiograms. However, applications other than quantitation of left ventricle structure and function exist for contrast enhanced left ventricular opacification. Contrast agents enable recording of Doppler velocity signals in patients with poor ultrasound transmission, providing estimates of aortic stenosis gradient and pulmonary artery pressures. Contrast echo is of value in detecting apical hypertrophic cardiomyopathy and accompanying apical aneurysms. Most importantly, ultrasound enhancing agents can identify apical and left atrial masses when they cannot be visualized in unenhanced images, and can distinguish thrombi from tumors by visualizing the vascularity inherent in tumors. Contrast agents distinguish trabecular from compacted myocardium in noncompaction syndrome, and hypertrabeculation with other abnormal conditions. A major potential application of ultrasound enhancing agents is myocardial opacification, which can assist in identifying nonviable myocardium. Also, the delayed reappearance of myocardial perfusion after microbubble destruction identifies impaired contrary flow and can diagnose coronary stenosis. Innovative applications of ultrasound contrast agents currently under investigation, include visualizing the vaso vasorum to identify plaques and assess their vulnerability, and theranostic agents to deliver drugs and biologists and to assist in sonothrombolysis. It is anticipated that the role of ultrasound contrast agents will continue to increase in the future.     

Single-nanometer iron oxide nanoparticles as tissue-permeable MRI contrast agents

Magnetic nanoparticles are robust contrast agents for MRI and often produce particularly strong signal changes per particle. Leveraging these effects to probe cellular- and molecular-level phenomena in tissue can, however, be hindered by the large sizes of typical nanoparticle contrast agents. To address this limitation, we introduce single-nanometer iron oxide (SNIO) particles that exhibit superparamagnetic properties in conjunction with hydrodynamic diameters comparable to small, highly diffusible imaging agents. These particles efficiently brighten the signal in T₁-weighted MRI, producing per-molecule longitudinal relaxation enhancements over 10 times greater than conventional gadolinium-based contrast agents. We show that SNIOs permeate biological tissue effectively following injection into brain parenchyma or cerebrospinal fluid. We also demonstrate that SNIOs readily enter the brain following ultrasound-induced blood–brain barrier disruption, emulating the performance of a gadolinium agent and providing a basis for future biomedical applications. These results thus demonstrate a platform for MRI probe development that combines advantages of small-molecule imaging agents with the potency of nanoscale materials.

Magnetic nanoparticles can dramatically shorten longitudinal and transverse relaxation times (T₁ and T₂) in MRI, and they consequently produce strong contrast in relaxation-weighted MRI scans (1, 2). There has been considerable interest in harnessing these effects for noninvasive assessment of physiological parameters in living subjects. Magnetic nanoparticles (3) have been functionalized to bind or sense a variety of biologically interesting targets (4), but the large size of most nanoparticles limits their ability to access and probe cellular-level phenomena in tissue. To address this problem, we undertook to create magnetic nanoparticles small enough to effectively permeate parenchyma while still exhibiting high magnetic moments with respect to traditional gadolinium-based MRI contrast agents. We reasoned that such particles could function as an attractive platform for molecular and cellular imaging applications.To facilitate tissue permeability , nanoparticles should have a size comparable to paramagnetic agents like gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA), which has a molecular diameter of about 1 nm. This would require synthesizing iron oxide–based contrast agents with diameters considerably lower than the smallest previously reported species (5, 6). To accomplish this, we sought to create magnetic single-nanometer iron oxide (SNIO) contrast agents by improving our recently reported magnetic nanoparticle synthesis procedures (6).     

Aligned carbon nanotubes as polarization-sensitive,molecular near-field detectors

Near-field scanning optical microscopes are widely used in imaging of subwavelength features in various material systems and nanostructures. For a variety of applications, polarization-sensitive near-field probes can provide valuable information on the nature and symmetry of the imaged nanoparticles and emitters. Conventional near-field optical microscopy lacks in-plane polarization sensitivity. Here, we use aligned single-wall carbon nanotubes as polarization-sensitive molecular scale probes to image the transverse near-field components of an optical Hertzian dipole antenna. Because of the Raman “antenna effect” in carbon nanotubes, only the near-field components along the nanotube axis are detected. These findings demonstrate that aligned carbon nanotubes can be used as polarization-sensitive near-field detectors.     

Adverse reactions to ultrasound contrast agents: is the risk worth the benefit?

The introduction of ultrasound contrast agents has led to a marked improvement in diagnostic capabilities in echocardiography. As no serious adverse events were seen during the preclinical development phase, ultrasound contrast agents were thought to be safe. Recently, three fatal and 19 severe, non-fatal adverse reactions were reported in a post marketing analysis of more than 150,000 studies of Sonovue, which has led to the addition of several contra-indications for the use of this ultrasound contrast agent. Although a strong relationship was established between the non-fatal cases and administration of Sonovue, a causal relationship between the fatal cases and the use of Sonovue is debatable. Therefore, the risk associated with the use of this ultrasound contrast agent should be judged carefully, taking into consideration the prevalence of adverse effects of other contrast media and diagnostic procedures used in cardiology.     

Optimization of ultrasound-mediated gene transfer: comparison of contrast agents and ultrasound modalities.

AIMS: Ultrasound (US)-enhanced gene transfer for cardiovascular disease is an emerging technique with translational relevance. Prior to pre-clinical applications, optimization of gene transfer using various US contrast agents and parameters is required. In order to do so, two clinically relevant contrast agents (Optison and PESDA), and two US modalities (dedicated continuous wave system and diagnostic scanner) were tested in vitro and in vivo. METHODS AND RESULTS: In vitro, luciferase activity was measured after exposure of primary vascular cells to combinations of luciferase plasmid, contrast agents, and US exposures. US gene transfer was consistently superior to controls. PESDA was better than Optison; there was no significant difference between US modalities. In vivo, luciferase activity in skeletal muscle of rats was measured after injection of plasmid or adenovirus, expressing luciferase with or without US exposure. Diagnostic US was superior to continuous wave. US plasmid gene transfer was highly localized, and was superior to all controls except adenovirus which lacked spatial specificity. To deliver a secreted transgene product, US gene transfer of a plasmid expressing tissue factor pathway inhibitor (TFPI) to skeletal muscle resulted in a dose-related increase in plasma activity for up to 5 days after delivery. CONCLUSION: US-enhanced plasmid gene transfer is capable of transducing skeletal muscle in vivo either directly or via an intravascular route. This enhanced nonviral method is an alternative to plasmid DNA alone or viral vectors.     

Role of microbubble ultrasound contrast agents in the non-invasive assessment of chronic hepatitis C-related liver disease

Patients who are chronically infected with the hepatitis C virus often develop chronic liver disease and assessment of the severity of liver injury is required prior to considering viral eradication therapy. This article examines the various assessment methods currently available from gold standard liver biopsy to serological markers and imaging. Ultrasound is one of the most widely used imaging modalities in clinical practice and is already a first-line diagnostic tool for liver disease. Microbubble ultrasound contrast agents allow higher resolution images to be obtained and functional assessments of microvas-cular change to be carried out. The role of these agents in quantifying the state of hepatic injury is discussed as a viable method of determining the stage and grade of liver disease in patients with hepatitis C. Although currently confined to specialist centres, the availability of microbubble contrast-enhanced ultrasound will inevitably increase in the clinical setting.     

载紫杉醇超声微泡造影剂对人肝癌HepG2细胞周期及形态的影响

目的 探讨超声辐照紫杉醇微泡造影剂,对人肝癌细胞株HepG2细胞周期的影响和形态学变化.方法 体外培养人肝癌HepG2细胞,将细胞分4组,即空白对照组,紫杉醇组,超声空白微泡组,超声载紫杉醇微泡组.流式细胞仪检测不同处理组细胞周期分布,透射电镜观察不同处理组形态学变化. 结果 超声载紫杉醇微泡组细胞阻滞在G2/M期;超声载紫杉醇微泡能够诱导肿瘤细胞发生凋亡,并有凋亡小体形成.结论 超声辐照载紫杉醇微泡造影剂对人肝癌细胞株HepG2有明显阻滞作用,并诱导肿瘤细胞发生凋亡.     

Evaluation of contrast agents for delineation of vessel wall boundary by intracoronary ultrasound after coronary angioplasty in human

We evaluated the potential for improving visualization at intervention sites using contrast-enhanced intracoronary ultrasound (ICUS) and the suitable contrast agents for this procedure in humans. In 37 patients, ICUS (30 MHz) was performed with intracoronary bolus injection (3 mL) of seven different contrast preparations and without the contrast agents (control) after coronary intervention. The contrast agents used were as follows: saline solution, standard iomeprol, standard ioxaglate, sonicated iomeprol, sonicated ioxaglate, 50% Albunex, and 100% Albunex. Homogeneous and complete opacification of the vessel lumen and false lumen was observed with sonicated ioxaglate, 50% and 100% Albunex. Shadowing was not observed at all with sonicated ioxaglate and was uncommon with 50% Albunex, whereas 100% Albunex caused shadowing in all cases. The coronary delineation rate with the other contrast agents was only 60%–70%, and the homogeneity and peak intensity were relatively low. Thus, sonicated ioxaglate and 50% Albunex both achieved good visualization, but the latter is more expensive, more difficult to handle, and takes longer to prepare. Of the agents we studied, sonicated ioxaglate appears to be best suited for contrast-enhanced ICUS. ICUS using suitable contrast agents could only visualize the large dissections and the strategy was changed according to the contrast-enhanced ICUS results in five cases. Thus, suitable contrast agents, e.g., sonicated ioxaglate, should be used during ICUS after intracoronary intervention.Cathet. Cardiovasc. Intervent. 47:6–13, 1999. © 1999 Wiley-Liss, Inc.     

Intravascular ultrasound complements the diagnostic capability of carbon dioxide digital subtraction angiography for patients with allergies to iodinated contrast medium

Carbon dioxide digital subtraction angiography (CO₂ DSA) is a useful and safe alternative for patients with renal dysfunction or allergies to iodinated contrast medium. However, CO₂ DSA image quality is worse than that of angiography with iodinated contrast medium, primarily because of movement during imaging and stent struts. In angioplasty of arteries of the lower extremities, CO₂ DSA cannot be used to sufficiently evaluate target lesions and determine the most efficient intervention. However, in the current case report, we describe a patient with severe allergies to iodinated contrast medium (Stevens–Johnson syndrome), because of which we were unable to use any iodinated contrast medium when conducting angioplasty. Therefore, we used intravascular ultrasound (IVUS), which facilitated the complete observation of the target lesion after stent implantation without requiring iodinated contrast medium. In this case, IVUS was used to complement the diagnostic capabilities of CO₂ DSA. © 2012 Wiley Periodicals, Inc.     

Phase III multicenter trial comparing the efficacy of 2% dodecafluoropentane emulsion (EchoGen) and sonicated 5% human albumin (Albunex) as ultrasound contrast agents in patients with suboptimal echocardiograms

Objectives. This study was performed to compare the safety and efficacy of intravenous 2% dodecafluoropentane (DDFP) emulsion (EchoGen) with that of active control (sonicated human albumin [Albunex]) for left ventricular (LV) cavity opacification in adult patients with a suboptimal echocardiogram.Background. The development of new fluorocarbon-based echocardiographic contrast agents such as DDFP has allowed opacification of the left ventricle after peripheral venous injection. We hypothesized that DDFP was clinically superior to the Food and Drug Administration–approved active control.Methods. This was a Phase III, multicenter, single-blind, active controlled trial. Sequential intravenous injections of active control and DDFP were given 30 min apart to 254 patients with a suboptimal echocardiogram, defined as one in which the endocardial borders were not visible in at least two segments in either the apical two- or four-chamber views. Studies were interpreted in blinded manner by two readers and the investigators.Results. Full or intermediate LV cavity opacification was more frequently observed after DDFP than after active control (78% vs. 31% for reader A; 69% vs. 34% for reader B; 83% vs. 55% for the investigators, p < 0.0001). LV cavity opacification scores were higher with DDFP (2.0 to 2.5 vs. 1.1 to 1.5, p < 0.0001). Endocardial border delineation was improved by DDFP in 88% of patients versus 45% with active control (p < 0.001). Similar improvement was seen for duration of contrast effect, salvage of suboptimal echocardiograms, diagnostic confidence and potential to affect patient management. There was no difference between agents in the number of patients with adverse events attributed to the test agent (9% for DDFP vs. 6% for active control, p = 0.92).Conclusions. This Phase III multicenter trial demonstrates that DDFP is superior to sonicated human albumin for LV cavity opacification, endocardial border definition, duration of effect, salvage of suboptimal echocardiograms, diagnostic confidence and potential to influence patient management. The two agents had similar safety profiles.