Scientists developed nanoparticles that respond to warmth inside tumors to boost ultrasound clarity, offering a safer and more targeted imaging method.
(Nanowerk Spotlight) Ultrasound imaging is a widely used method for diagnosing cancer. It is noninvasive, fast, and offers real-time visualization, but its effectiveness is often limited by the performance of the contrast agents used to enhance image clarity. The most common agents today are gas-filled microbubbles. These particles, while useful for vascular imaging, are relatively large—typically between one and five micrometers in diameter. Their size prevents them from leaving the bloodstream and entering tumor tissues, where imaging is most needed. As a result, they are cleared quickly from circulation and contribute little to long-term imaging of solid tumors.
Efforts to address this limitation have led researchers to explore nanoscale alternatives. Particles under 700 nanometers can accumulate at tumor sites by exploiting a property of tumor blood vessels called the enhanced permeability and retention (EPR) effect. These vessels are leaky and have poor drainage, allowing small particles to pass through and build up in the surrounding tumor tissue.
Some recent approaches have focused on encapsulating phase-change materials into nanocarriers. These materials can shift from one physical state to another—such as forming tiny droplets—when exposed to heat, ultrasound, or light. These changes alter the way sound waves reflect off them, generating stronger signals in ultrasound images.
Biological carriers such as extracellular vesicles offer another avenue. These are small membrane-bound structures naturally released by cells. Among them, bacterial outer membrane vesicles (OMVs) are of particular interest. Produced by Gram-negative bacteria such as Escherichia coli, OMVs measure about 100 nanometers and contain proteins and lipids from the bacterial outer membrane. OMVs can accumulate in tumors and have been shown to influence immune responses. Unlike synthetic nanoparticles, they exhibit high biocompatibility and are more adaptable to molecular modifications.
OMVs have previously been explored for fluorescence and photoacoustic imaging, but their use in ultrasound has remained limited. A central obstacle is that their internal structure lacks the phase boundaries needed to reflect ultrasound waves effectively.
Preparation and characterization of nB-OMVs. A) Schematic illustration to show the preparation process of nB-OMVs. B) GC-MS Analysis of OMVs and nB-OMVs. C) TEM images of OMVs and nB-OMVs. Scale bars: 600 nm. D,E) Nanoparticle tracking analysis and mean particle size of OMVs and nB-OMVs. F) Zeta potential of OMVs and nB-OMVs by DLS analysis. G) Coomassie blue staining of OMVs and nB-OMVs by SDS-PAGE. H) Western blot analysis was obtained for the presence of two characteristic proteins of OMVs and nB-OMVs. Data are presented as mean ± SD (n = 3). (Image: Reprinted with permission by Wiley-VCH Verlag) (click on image to enlarge)
The OMVs were isolated from E. coli cultures and then mixed with n-butanol under agitation. This process allowed n-butanol to insert into the lipid bilayer of the vesicles without disrupting their structure. Electron microscopy confirmed that the OMVs retained their spherical form after loading. Nanoparticle tracking showed only a slight increase in average diameter. Surface charge measurements and protein analysis indicated that nB-OMVs preserved the key properties of native OMVs, including their membrane composition and dispersion stability.
At room temperature, both OMVs and nB-OMVs produced negligible ultrasound signals. However, when heated to 45 degrees Celsius, nB-OMVs generated strong echoes. This is explained by the behavior of n-butanol in water: as temperature rises, its solubility decreases, leading to the formation of internal droplets within the vesicles. These droplets introduce new boundaries that reflect ultrasound waves and enhance image contrast.
Further experiments confirmed this mechanism. A mixture of water and n-butanol produced echoes when heated, as did model lipid vesicles loaded with n-butanol. Control tests with alcohols that fully dissolve in water—such as ethanol and methanol—showed no effect. Only alcohols with low water solubility, including n-butanol, n-pentanol, and n-hexanol, generated detectable echoes. This highlighted the necessity of phase separation for acoustic signal generation.
Frequency analysis of the ultrasound signals showed that nB-OMVs produced not only fundamental echoes but also harmonic and subharmonic components. These nonlinear signals are critical for contrast-enhanced ultrasound (CEUS), which uses harmonic reflections to improve imaging accuracy. Increasing the concentration of n-butanol during preparation led to stronger signals. The effect was stable over a ten-minute heating period, long enough to support real-time imaging applications.
To evaluate how nB-OMVs behave in living systems, the researchers injected fluorescently labeled vesicles into mice with pancreatic tumors. Both nB-OMVs and unmodified OMVs accumulated in tumors within six hours. Imaging and tissue analysis showed that nB-OMVs successfully crossed blood vessel walls and entered tumor cells. The addition of n-butanol did not impair these properties. Tumor growth was modestly suppressed in both groups, consistent with the known bioactivity of OMVs, which carry bacterial membrane proteins capable of immune modulation and direct tumor cell interaction.
A unique feature of OMVs is their ability to induce red blood cell (RBC) leakage into tumors. This is likely due to the presence of lipopolysaccharides (LPS) on the vesicle surface, which can disrupt vascular integrity. The study used this feature to enable targeted heating. Hemoglobin in the extravasated RBCs absorbs near-infrared (NIR) light and converts it into heat. After injecting nB-OMVs, the researchers applied an 808-nanometer NIR laser to the tumor site. This caused a localized rise in temperature, triggering phase separation of n-butanol inside the nB-OMVs and significantly boosting ultrasound signal.
Ultrasound scans taken after laser exposure showed clear differences between groups. Mice treated with OMVs showed no increase in image brightness, but those injected with nB-OMVs displayed progressively stronger signals with each minute of heating. After eight minutes, the signal in B-mode increased by 4.6 times, while CEUS mode saw an 8.5-fold enhancement. Measurements of signal-to-noise and contrast-to-tissue ratios confirmed the effect. These findings established that nB-OMVs can be selectively activated at tumor sites to enhance imaging contrast.
The study also examined safety. The amount of n-butanol delivered per injection was well below known toxic thresholds. Hemolysis tests confirmed low impact on red blood cells. A mild and temporary increase in liver enzymes and white blood cell counts was observed, indicating a short-lived immune response. No organ damage was detected by histology. Body weight returned to normal within a few days, and blood parameters remained within reference ranges over the 28-day observation period. These results support the biosafety of nB-OMVs for short-term diagnostic use.
This work presents a strategy for converting naturally derived vesicles into functional ultrasound contrast agents through a simple, heat-sensitive modification. The nB-OMVs retain the tumor-targeting behavior of OMVs while adding a thermally controlled imaging capability. Their small size allows for deep tissue penetration, and their biological origin offers advantages in compatibility and engineering flexibility.
While the current design requires near-infrared light for activation—a method limited by shallow tissue penetration—alternative heating approaches such as focused ultrasound or endoscopic light delivery could extend its clinical relevance.
The study demonstrates how the combination of bacterial vesicle biology with simple chemical modifications can create responsive diagnostic tools. By integrating selective tumor accumulation, local activation, and ultrasound visibility, nB-OMVs represent a step toward multifunctional agents for real-time imaging. The approach could be adapted to other vesicle types and potentially combined with therapeutic payloads, making it a platform for both diagnosis and treatment in future applications.
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