Nanomaterials incorporated ultrasound contrast agents for cancer theranostics

Nanotechnology provides various nanomaterials with tremendous functionalities for cancer diagnostics and therapeutics.Recently, theranostics has been developed as an alternative strategy for efficient cancer treatment through combination of imaging diagnosis and therapeutic interventions under the guidance of diagnostic results. Ultrasound(US) imaging shows unique advantages with excellent features of real-time imaging, low cost, high safety and portability, making US contrast agents(UCAs)an ideal platform for construction of cancer theranostic agents. This review focuses on the development of nanomaterials incorporated multifunctional UCAs serving as theranostic agents for cancer diagnostics and therapeutics, via conjugation of superparamagnetic iron oxide nanoparticles(SPIOs), Cu S nanoparticles, DNA, si RNA, gold nanoparticles(GNPs), gold nanorods(GNRs), gold nanoshell(GNS), graphene oxides(GOs), polypyrrole(PPy) nanocapsules, Prussian blue(PB) nanoparticles and so on to different types of UCAs. The cancer treatment could be more effectively and accurately carried out under the guidance and monitoring with the help of the achieved theranostic agents. Furthermore, nanomaterials incorporated theranostic agents based on UCAs can be designed and constructed by demand for personalized and accurate treatment of cancer, demonstrating their great potential to address the challenges of cancer heterogeneity and adaptation, which can provide alternative strategies for cancer diagnosis and therapeutics.

Ultrasound contrast agents from microbubbles to biogenic gas vesicles

Microbubbles have been the earliest and most widely used ultrasound contrast agents by virtue of their unique features:such as non-toxicity,intravenous inject-ability,ability to cross the pulmonary capillary bed,and significant enhancement of echo signals for the duration of the examination,resulting in essential preclinical and clinical applications.The use of microbubbles functional-ized with targeting ligands to bind to specific targets in the bloodstream has further enabled ultrasound molecular imaging.Nevertheless,it is very challenging to utilize targeted microbubbles for molecular imaging of extra-vascular targets due to their size.A series of acoustic nanomaterials have been developed for breaking free from this constraint.Especially,biogenic gas vesicles,gas-filled protein nanostructures from microorganisms,were engineered as thefirst biomolecular ultrasound contrast agents,opening the door for more direct visual-ization of cellular and molecular function by ultrasound imaging.The ordered protein shell structure and unique gasfilling mechanism of biogenic gas vesicles endow them with excellent stability and attractive acoustic responses.What’s more,their genetic encodability enables them to act as acoustic reporter genes.This article reviews the upgrading progresses of ultrasound contrast agents from microbubbles to biogenic gas vesicles,and the opportu-nities and challenges for the commercial and clinical translation of the nascentfield of biomolecular ultrasound.

Characterization of the interaction of nanobubble ultrasound contrast agents with human blood components

Nanoscale ultrasound contrast agents,or nanobubbles,are being explored in preclinical applications ranging from vascular and cardiac imaging to targeted drug delivery in cancer.These sub-micron particles are approximately 10x smaller than clinically available microbubbles.This allows them to effectively traverse compromised physiological barriers and circulate for extended periods of time.While various aspects of nanobubble behavior have been previously examined,their behavior in human whole blood has not yet been explored.Accordingly,herein we examined,for the first time,the short and long-term effects of blood components on nanobubble acoustic response.We observed differences in the kinetics of backscatter from nanobubble suspensions in whole blood compared to bubbles in phosphate buffered saline(PBS),plasma,or red blood cell solutions(RBCs).Specifically,after introducing nanobubbles to fresh human whole blood,signal enhancement,or the magnitude of nonlinear ultrasound signal,gradually increased by 22.8±13.1%throughout our experiment,with peak intensity reached within 145 s.In contrast,nanobubbles in PBS had a stable signal with negligible change in intensity(1.7±3.2%)over 8 min.Under the same conditions,microbubbles made with the same lipid formulation showed a56.8±6.1%decrease in enhancement in whole blood.Subsequent confocal,fluorescent,and scanning electron microscopy analysis revealed attachment of the nanobubbles to the surface of RBCs,suggesting that direct interactions,or hitchhiking,of nanobubbles on RBCs in the presence of plasma may be a possible mechanism for the observed effects.This phenomenon could be key to extending nanobubble circulation time and has broad implications in drug delivery,where RBC interaction with nanoparticles could be exploited to improve delivery efficiency.

Role for contrast-enhanced ultrasound in assessing complications after kidney transplant

Kidney transplantation(KT)is an effective treatment for end-stage renal disease.Despite their rate has reduced over time,post-transplant complications still represent a major clinical problem because of the associated risk of graft failure and loss.Thus,post-KT complications should be diagnosed and treated promptly.Imaging plays a pivotal role in this setting.Grayscale ultrasound(US)with color Doppler analysis is the first-line imaging modality for assessing complications,although many findings lack specificity.When performed by experienced operators,contrast-enhanced US(CEUS)has been advocated as a safe and fast tool to improve the accuracy of US.Also,when performing CEUS there is potentially no need for further imaging,such as contrast-enhanced computed tomography or magnetic resonance imaging,which are often contraindicated in recipients with impaired renal function.This technique is also portable to patients’bedside,thus having the potential of maximizing the cost-effectiveness of the whole diagnostic process.Finally,the use of blood-pool contrast agents allows translating information on graft microvasculature into time-intensity curves,and in turn quantitative perfusion indexes.Quantitative analysis is under evaluation as a tool to diagnose rejection or other causes of graft dysfunction.In this paper,we review and illustrate the indications to CEUS in the post-KT setting,as well as the main CEUS findings that can help establishing the diagnosis and planning the most adequate treatment.

Nonlinear response of ultrasound contrast agent microbubbles:From fundamentals to applications

Modelling and biomedical applications of ultrasound contrast agent （UCA） microbubbles have attracted a great deal of attention. In this review, we summarize a series of researches done in our group, including （i） the development of an all-in-one solution of characterizing coated bubble parameters based on the light scattering technique and flow cytometry; （ii） a novel bubble dynamic model that takes into consideration both nonlinear shell elasticity and viscosity to eliminate the dependences of bubble shell parameters on bubble size; （iii） the evaluation of UCA inertial cavitation threshold and its relationship with shell parameters; and （iv） the investigations of transfection efficiency and the reduction of cytotoxicity in gene delivery facilitated by UCAs excited by ultrasound exposures.

Enhanced endoscopic ultrasound imaging for pancreatic lesions: The road to artificial intelligence

Early detection of pancreatic cancer has long eluded clinicians because of its insidious nature and onset.Often metastatic or locally invasive when symptomatic,most patients are deemed inoperable.In those who are symptomatic,multi-modal imaging modalities evaluate and confirm pancreatic ductal adenocarcinoma.In asymptomatic patients,detected pancreatic lesions can be either solid or cystic.The clinical implications of identifying small asymptomatic solid pancreatic lesions(SPLs)of<2 cm are tantamount to a better outcome.The accurate detection of SPLs undoubtedly promotes higher life expectancy when resected early,driving the development of existing imaging tools while promoting more comprehensive screening programs.An imaging tool that has matured in its reiterations and received many image-enhancing adjuncts is endoscopic ultrasound(EUS).It carries significant importance when risk stratifying cystic lesions and has substantial diagnostic value when combined with fine needle aspiration/biopsy(FNA/FNB).Adjuncts to EUS imaging include contrast-enhanced harmonic EUS and EUS-elastography,both having improved the specificity of FNA and FNB.This review intends to compile all existing enhancement modalities and explore ongoing research around the most promising of all adjuncts in the field of EUS imaging,artificial intelligence.

Ultrasound-mediated transdermal drug delivery of fluorescent nanoparticles and hyaluronic acid into porcine skin in vitro

Transdermal drug delivery （TDD） can effectively bypass the first-pass effect. In this paper, ultrasound-facilitated TDD on fresh porcine skin was studied under various acoustic parameters, including frequency, amplitude, and exposure time. The delivery of yellow-green fluorescent nanoparticles and high molecular weight hyaluronic acid （HA） in the skin samples was observed by laser confocal microscopy and ultraviolet spectrometry, respectively. The results showed that, with the application of ultrasound exposures, the permeability of the skin to these markers （e.g., their penetration depth and concentration） could be raised above its passive diffusion permeability. Moreover, ultrasound-facilitated TDD was also tested with/without the presence of ultrasound contrast agents （UCAs）. When the ultrasound was applied without UCAs, low ultrasound frequency will give a better drug delivery effect than high frequency, but the penetration depth was less likely to exceed 200 p.m. However, with the help of the ultrasound-induced microbubble cavitation effect, both the penetration depth and concentration in the skin were significantly enhanced even more. The best ultrasound-facilitated TDD could be achieved with a drug penetration depth of over 600 p.m, and the penetration concentrations of fluorescent nanoparticles and HA increased up to about 4-5 folds. In order to get better understanding of ultrasound-facilitated TDD, scanning electron microscopy was used to examine the surface morphology of skin samples, which showed that the skin structure changed greatly under the treatment of ultrasound and UCA. The present work suggests that, for TDD applications （e.g., nanoparticle drug carriers, transdermal patches and cosmetics）, protocols and methods presented in this paper are potentially useful.

In vivo transfection of enhanced green fluorescent protein in rat retinal ganglion cells mediated by ultrasound-induced microbubbles

BACKGROUND： Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein （EGFP） in in vitro cultured retinal ganglial cells （RGCs）. OBJECTIVE： To investigate the feasibility of ultrasound-mediated microbubble destruction for EGFP transfection in rat RGCs, and to compare efficiency and cell damage with traditional transfection methods. DESIGN, TIME AND SETTING： In vivo, gene engineering experiment. The study was performed at the Central Laboratory, Institute of Ultrasonic Imaging, Chongqing Medical University from March to July 2008. MATERIALS： Eukaryotic expression vector plasmid EGFP and microbubbles were prepared by the Institute of Ultrasonic Imaging, Chongqing Medical University. The microbubbles were produced at a concentration of 8.7 × 10^11/L, with a 2-4 μm diameter, and 10-hour half-life in vitro. METHODS： A total of 50 Sprague Dawley rats were randomly assigned to four groups. Normal controls （n = 5） were infused with 5 μL normal saline to the vitreous cavity; the naked plasmid group （n = 15） was infused with 5 pL EGFP plasmid to the vitreous cavity; in the plasmid with ultrasound group （n = 15）, the eyes were irradiated with low-energy ultrasound wave （0.5 W/cm^2） for a total of 60 seconds （irradiated for 5 seconds, at 10-second intervals） immediately following infusion of EGFP plasmids to the vitreous cavities. In the microbubble-ultrasound group （n = 15）, the eyes were irradiated with the same power of ultrasonic wave immediately following infusion of microbubbles containing EGFP plasmids to the vitreous cavities. MAIN OUTCOME MEASURES： After 7 days, retinal preparations and EGFP expression in RGCs were observed by fluorescence microscopy. RGC quantification in the retinal ganglion cell layer was performed. In addition, EGFP mRNA expression was semi-quantitatively determined by RT-PCR. RESULTS： The transfection efficiency of EGFP to RGCs by microbubbles with ultrasound was significantly greater than the other groups, and no obvious damage was detected in the RGCs. CONCLUSION： Under irradiation of low-frequency ultrasound waves, ultrasound-mediated microbubble destruction was effective and resulted in safe transfection of the EGFP gene to the RGCs.

Advantages of contrast-enhanced ultrasound in the localization and diagnostics of sentinel lymph nodes in breast cancer

Sentinel lymph nodes(SLNs)are the first station of lymph nodes that extend from the breast tumor to the axillary lymphatic drainage.The pathological status of these LNs can predict that of the entire axillary lymph node.Therefore,the accurate identification of SLNs is necessary for sentinel lymph node biopsy(SLNB)to replace axillary lymph node dissection(ALND).The quality of life and prognosis of breast cancer patients are related to proper surgical treatment after the precise identification of SLNs.Some of the SLN tracers that have been identified include radioisotope,nano-carbon,indocyanine green(ICG),and methylene blue(MB).However,these tracers have certain limitations,such as pigmentation,radiation dangers,and the requirement for costly detection equipment.Ultrasound contrast agents(UCAs)have good specificity and sensitivity,and thus can compensate for some shortcomings of the mentioned tracers.This technique is also being applied to SLNB in patients with breast cancer,and can even provide an initial judgment on SLN status.Contrast-enhanced ultrasound(CEUS)has the advantages of high distinguishability,simple operation,no radiation harm,low cost,and accurate localization;therefore,it is expected to replace the traditional biopsy methods.In addition,it can significantly enhance the accuracy of SLN localization and shorten the operation time.

Subharmonic scattering of ultrasound contrast agent microbubbles may be an effective and promising tool for portal vein pressure estimation

Portal hypertension(PH)is a commonly observed syndrome in patients with cirrhosis and other chronic liver diseases(1).It is closely associated with severe clinical complications,including upper gastrointestinal hemorrhage,ascites,hepatic encephalopathy,and liver failure(2-6).Portal vein pressure(PVP)is a prognostic indicator for patients with cirrhosis,and PH is a contraindication for hepatectomy(7,8).Currently,the gold standard for assessing PVP is the measurement of PVP gradient[hepatic venous pressure gradient(HVPG)],which is indirectly determined by placing a catheter in the hepatic vein(3).

Off-label-use of sulfur-hexafluoride in voiding urosonography for diagnosis of vesicoureteral reflux in children:A survey on adverse events

AIM To evaluate the risk profile of sulfur hexafluoride in voiding urosonography(VUS) based on a large cohort of children.METHODS Since 2011 sulfur hexafluoride(SH,SonoV ue?,Bracco,Italy) is the only ultrasound contrast available in the European Union and its use in children has not been approved.Within a 4-year-period,531 children with suspected or proven vesicoureteral reflux(f/m = 478/53; mean age 4.9 years; 1 mo-25.2 years) following parental informed consent underwent VUS with administration of 2.6 ± 1.2 mL SH in a two-center study.A standardizedtelephone survey on adverse events was conducted three days later.RESULTS No acute adverse reactions were observed.The survey revealed subacute,mostly self-limited adverse events in 4.1%(22/531).The majority of observed adverse events(17/22) was not suspected to be caused by an allergic reaction: Five were related to catheter placement,three to reactivated urinary tract infections,five were associated with perineal disinfection before voiding urosonography or perineal dermatitis and four with a common cold.In five patients(0.9%) hints to a potential allergic cause were noted: Perineal urticaria was reported in three interviews and isolated,mild fever in two.These were minor self-limited adverse events with a subacute onset and no hospital admittance was necessary.Ninety-six point two percent of the parents would prefer future VUS examinations with use of SH.CONCLUSION No severe adverse events were observed and indications of self-limited minor allergic reactions related to intravesical administration of SH were reported in less than 1%.

Interaction between encapsulated microbubbles:A finite element modelling study

Theoretical studies on the multi-bubble interaction are crucial for the in-depth understanding of the mechanism behind the applications of ultrasound contrast agents （UCAs） in clinics. A two-dimensional （2D） axisymmetric finite element model （FEM） is developed here to investigate the bubble-bubble interactions for UCAs in a fluidic environment. The effect of the driving frequency and the bubble size on the bubble interaction tendency （viz., bubbles＇ attraction and repulsion）, as well as the influences of bubble shell mechanical parameters （viz., surface tension coefficient and viscosity coefficient） are discussed. Based on FEM simulations, the temporal evolution of the bubbles＇ radii, the bubble-bubble distance, and the distribution of the velocity field in the surrounding fluid are investigated in detail. The results suggest that for the interacting bubble-bubble couple, the overall translational tendency should be determined by the relationship between the driving frequency and their resonance frequencies. When the driving frequency falls between the resonance frequencies of two bubbles with different sizes, they will repel each other, otherwise they will attract each other. For constant acoustic driving parameters used in this paper, the changing rate of the bubble radius decreases as the viscosity coefficient increases, and increases first then decreases as the bubble shell surface tension coefficient increases, which means that the strength of bubble-bubble interaction could be adjusted by changing the bubble shell visco-elasticity coefficients. The current work should provide a powerful explanation for the accumulation observations in an experiment, and provide a fundamental theoretical support for the applications of UCAs in clinics.

Subharmonic aided pressure estimation in portal hypertension:A noninvasive ultrasound-based technique to assess portal pressure gradient

Portal hypertension(PH)is a clinical syndrome,characterized by elevated pressure gradient between portal vein and inferior vena cava.These elevated pressures gradient due to increased vascular resistance and/or increased volume of blood flowing through the portal vein circulation,results in blood outflow difficulty from portal vein to hepatic veins and inferior vena cava.

Diagnostic value of contrast-enhanced intraoperative Doppler sonography for cerebral arteriovenous malformations compared with angiography

Background Intraoperative Doppler sonography has been used in the neurosurgical operating room for the localization and description of cerebral arteriovenous malformations （AVMs）. This study aimed to investigate the clinical value of contrast-enhanced intraoperative Doppler sonography, including its ability to assess the location and identify of feeding arteries in patients with AVMs and to compare this method with angiography.Methods Twenty-three patients with cerebral AVMs who were diagnosed using angiography, were examined with contrast-enhanced intraoperative Doppler sonography. As an echo-enhancing agent, Sulphur Hexafluoride Microbubbles for Injection （＂SonoVue＂） was administered intravenously in all patients. Sonogram results were reviewed and correlated with angiographic findings. For statistical analysis, the Wilcoxon signed-rank test was applied.Results Angiography identified 20 AVM lesions in the anterior or middle fossa and 3 in the posterior fossa.Contrast-enhanced intraoperative Doppler was somewhat less sensitive for only detecting 21/23 （91.3%） of the AVM lesions. Additionally, contrast-enhanced intraoperative Doppler slightly underestimated AVM size compared with angiographic findings but showed feeding arteries with sufficient acoustic properties. In 15 patients, angiography revealed a coincidental blood supply from another intracranial vessel, which was missed by contrast-enhanced intraoperative Doppler sonography.Conclusions In a limited group of patients with AVMs, contrast-enhanced intraoperative Doppler sonography was a less sensitive but useful and simple method for the detection of AVMs in contrast to angiography. No specific untoward effects were attributed to the use of ＂SonoVue＂ as a contrast-enhancing substance.