We report an ultrasound contrast agent for which we engineered the shell structure to impart much better stability under intense stress and deformation.
Contrast-enhanced ultrasound with microbubbles has shown promise in detection of prostate cancer (PCa), but sensitivity and specificity of detection remain challenging. Targeted nanoscale contrast agents with improved capability to accumulate in tumors may result in prolonged signal enhancement and improved detection of PCa with ultrasound. Here we report on a new nanobubble contrast agent that specifically targets prostate specific membrane antigen (PSMA) overexpressed in most prostate tumors. The PSMA-targeted bubbles (PSMA-NB) were utilized to simultaneously image dual flank PCa tumors (PSMA-positive PC3pip and PSMA-negative PC3flu) to examine whether the biomarker can be successfully detected and imaged using this probe in a mouse model. Results demonstrate that active targeting of NBs to PSMA rapidly and selectively enhances tumor accumulation and is critical for tumor retention of the contrast agent. Importantly, these processes could be visualized and quantified, in real time, with standard clinical ultrasound. Such demonstration of the immense yet underutilized potential of ultrasound in the area of molecular imaging can open the door to future opportunities for improving sensitivity and specificity of cancer detection using parametric NB-enhanced ultrasound imaging.Despite significant efforts, prostate cancer (PCa) is still the second most common leading cause of cancer-related deaths worldwide, with 180,000 new cases diagnosed in the USA in 2018 [1][2] . Accurate diagnosis of PCa is a crucial step necessary for informing the clinical management of the disease, yet conventional options leave much space for improvement. Currently, men with an abnormal digital rectal exam and/or increased levels of prostate serum antigen (PSA) are considered at high risk for cancer and are referred for a prostate biopsy to assess if PCa is present.The standard PCa biopsy procedure uses transrectal ultrasound (US) guidance to determine the prostate gland orientation, but the delineation of tumors within the prostate using US is unclear. Accordingly, biopsies are performed in a systematic manner by selecting 6-12 or more area from the peripheral zone of the prostate. These cores represent only 1% of prostate tissue and are a gross under sampling of prostate gland tissue, and biopsies performed using this conventional procedure result in significant false negatives of up to 50% [3][4][5] . Concern over the lack of pathological data in
A resonant mass measurement technique simultaneously distinguishes and characterizes (size and concentration) buoyant and non-buoyant particles in a bubble sample.
Ultrasound (US) is
a widely used diagnostic imaging tool because
it is inexpensive, safe, portable, and broadly accessible. Ultrasound
contrast agents (UCAs) are employed to enhance backscatter echo and
improve imaging contrast. The most frequently utilized UCAs are echogenic
bubbles made with a phospholipid or protein-stabilized hydrophobic
gas core. While clinically utilized, applications of UCAs are often
limited by rapid signal decay (<5 min) in vivo under typical ultrasound
imaging protocols. Here, we report on a formulation of lipid shell–stabilized
perfluoropropane (C3F8) microbubbles and nanobubbles
with a significantly prolonged in vivo stability. Microbubbles (875
± 280 nm) of the target size were prepared by utilizing a multiple-step
centrifugation cycle, while nanobubbles (299 ± 189 nm) were isolated
from the activated vial using a single centrifugation step. To provide
in-depth acoustic characterization of the new construct we evaluated
the effect of size and concentration on their in vitro and in vivo
performance. In vitro and in vivo characterization were carried out
for a range of bubble concentrations normalized by total gas volume
quantified via headspace gas chromatography/mass spectrometry (GC/MS).
In vitro characterization revealed that nanobubbles at different concentrations
are more consistently stable over time with the highest and lowest
dilutions (50-fold decrease) only differing in US signal after 8 min
exposure by 10.34%, while for microbubbles the difference was 86.46%.
As expected, due to the difference in hydrodynamic diameter and scattering
cross section difference, nanobubbles showed lower overall initial
signal intensity. In vivo experiments showed that both microbubbles
and nanobubbles with similar initial peak signal intensity are comparably
stable over time with 66.8% and 60.6% remaining signal after 30 min,
respectively. This study demonstrates that bubble concentration has
significant effects on the persistence of both microbubbles and nanobubbles
in vitro and in vivo, but the effects are more pronounced in larger
bubbles. These effects should be taken into account when selecting
the appropriate bubble parameters for future imaging applications.
In this study, we investigated the effect of positively and negatively charged Fe3O4 and TiO2 nanoparticles (NPs) on the growth of soybean plants (Glycine max.) and their root associated soil microbes. Soybean plants were grown in a greenhouse for six weeks after application of different amounts of NPs, and plant growth and nutrient content were examined. Roots were analyzed for colonization by arbuscular mycorrhizal (AM) fungi and nodule-forming nitrogen fixing bacteria using DNA-based techniques. We found that plant growth was significantly lower with the application of TiO2 as compared to Fe3O4 NPs. The leaf carbon was also marginally significant lower in plants treated with TiO2 NPs; however, leaf phosphorus was reduced in plants treated with Fe3O4. We found no effects of NP type, concentration, or charge on the community structure of either rhizobia or AM fungi colonizing plant roots. However, the charge of the Fe3O4 NPs affected both colonization of the root system by rhizobia as well as leaf phosphorus content. Our results indicate that the type of NP can affect plant growth and nutrient content in an agriculturally important crop species, and that the charge of these particles influences the colonization of the root system by nitrogen-fixing bacteria.
Understanding
the pressure dependence of the nonlinear behavior
of ultrasonically excited phospholipid-stabilized nanobubbles (NBs)
is important for optimizing ultrasound exposure parameters for implementations
of contrast enhanced ultrasound, critical to molecular imaging. The
viscoelastic properties of the shell can be controlled by the introduction
of membrane additives, such as propylene glycol as a membrane softener
or glycerol as a membrane stiffener. We report on the production of
high-yield NBs with narrow dispersity and different shell properties.
Through precise control over size and shell structure, we show how
these shell components interact with the phospholipid membrane, change
their structure, affect their viscoelastic properties, and consequently
change their acoustic response. A two-photon microscopy technique
through a polarity-sensitive fluorescent dye, C-laurdan, was utilized
to gain insights on the effect of membrane additives to the membrane
structure. We report how the shell stiffness of NBs affects the pressure
threshold (
P
t
) for the sudden amplification
in the scattered acoustic signal from NBs. For narrow size NBs with
200 nm mean size, we find
P
t
to be between
123 and 245 kPa for the NBs with the most flexible membrane as assessed
using C-Laurdan, 465–588 kPa for the NBs with intermediate
stiffness, and 588–710 kPa for the NBs with stiff membranes.
Numerical simulations of the NB dynamics are in good agreement with
the experimental observations, confirming the dependence of acoustic
response to shell properties, thereby substantiating further the development
in engineering the shell of ultrasound contrast agents. The viscoelastic-dependent
threshold behavior can be utilized for significantly and selectively
enhancing the diagnostic and therapeutic ultrasound applications of
potent narrow size NBs.
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