Biology subjects

Kolios, M. C.

Publications and source records attributed to Kolios, M. C..

7 recordsLinked to original sources

bioRxiv2026

Membrane Filtration Removes Lipids and Compromises the Acoustic Stability of Nanobubbles

Nanobubbles (NBs) have been widely investigated as ultrasound contrast and therapeutic agents, yet whether submicron gas bubbles can directly generate measurable ultrasound signals remains controversial. Because acoustic scattering scales with bubble size, several studies have recently attributed nanobubble echogenicity primarily to larger bubbles. A recent Science Advances publication, attributed the loss of ultrasound signal after filtration to the removal of residual larger bubbles [1]. This interpretation assumes that filtration acts solely as a size-selection process. Here, we show that filtration also alters the shell properties and stability of lipid-coated NBs, independent of size. Filtered NBs (F-NBs) show significant lipid loss and accumulation on the filters themselves, resulting in decreased bubble stability and greater susceptibility to destruction under acoustic excitation. To decouple size from processing effects, we generated NB populations size-matched to filtered samples using increased centrifugation (IC-NBs), a comparatively gentler separation method. Despite comparable size distributions, IC-NBs retained sustained ultrasound scattering, whereas filtered NBs showed significantly reduced acoustic persistence. Filtration through 5 m pores, ~5-fold larger than the largest bubbles, preserved acoustic persistence but still reduced bubble concentration by ~30%, suggesting that membrane contact may contribute to bubble loss beyond size exclusion. Complementary lipid and gas-content assays provided further evidence of filtration-induced changes. The 0.45 m filtration removed substantially more total lipid than could be attributed to free-lipid removal alone, while the lipid associated with the larger bubbles removed during filtration accounted for only ~5% of the additional lipid loss. Encapsulated gas content remained comparable to that of size-matched centrifuged controls, suggesting that the reduced acoustic stability of filtered NBs is more closely associated with changes in lipid content than with loss of the gas core. These results indicate that membrane filtration is not a purely size-selective process: it removes substantially more lipid than size exclusion predicts and yields bubbles that are less stable under ultrasound, most likely through changes in shell lipid content or in the surrounding lipid reservoir, underscoring the importance of processing-induced effects in interpreting nanobubble echogenicity.

biophysics↗

bioRxiv2026

Metabolic and Anti-Proliferative Responses of Pancreatic Cancer Cells to Ultrasound and Nanobubble Treatment

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies due to its dense stroma, which limits drug delivery and therapeutic efficacy. Ultrasound (US) mediated strategies using nanobubbles (NBs) offer a promising approach to enhance treatment, yet the biological effects of NB exposure and the timing of US application remain unclear. Here, we investigated how NB exposure with immediate (0h) or delayed (1h) US affects viability, proliferation, metabolism, and stress signaling in PANC-1 and BxPC-3 cells. Immediate US exposure in the presence of extracellular nanobubbles resulted in a greater reduction in cell viability at 24 h compared to delayed US application. Proliferation analysis showed that Ki67 positivity decreased following USNB treatments in both cell lines. Metabolically, NB treatment alone increased cellular activity, whereas combined USNB treatment reduced metabolic activity over time. Seahorse analysis revealed higher basal respiration in PANC-1 cells compared to BxPC-3 cells, consistent with a more glycolytic phenotype, while USNB treatment enhanced glycolytic responses, particularly in PANC-1. Moreover, stress responses were also more pronounced in PANC-1 cells, with HSP70 expression increasing up to 2-fold in NB incubated group and decreasing in USNB groups compared to untreated, whereas BxPC-3 cells exhibited only modest and opposite changes to PANC-1 in HSP70 expression decreasing with NB incubation. Treatment timing critically influenced outcomes, with immediate US producing stronger antiproliferative and cytotoxic effects, highlighting the importance of sequencing in USNB therapeutic strategies. Moreover, NBs alone stimulated metabolic and stress responses that may promote proliferation, whereas NBs combined with US induced stronger stress responses associated with metabolic reprogramming and reduced proliferation.

cancer biology↗

bioRxiv2023

Assessing Tumor Microenvironment Characteristics and Stratifying EPR with a Nanobubble Companion Nanoparticle via Contrast-Enhanced Ultrasound Imaging

The tumor microenvironment is characterized by dysfunctional endothelial cells, resulting in heightened vascular permeability. Many nanoparticle-based drug delivery systems attempt to use this enhanced permeability combined with impaired lymphatic drainage (a concept known as the enhanced permeability and retention effect or EPR effect) as the primary strategy for drug delivery, but this has not proven to be as clinically effective as anticipated. The specific mechanisms behind the inconsistent clinical outcomes of nanotherapeutics have not been clearly articulated, and the field has been hampered by a lack of accessible tools to study EPR-associated phenomena in clinically relevant scenarios. While medical imaging has tremendous potential to contribute to this area, it has not been broadly explored. This work examines, for the first time, the use of multiparametric dynamic contrast-enhanced ultrasound (CEUS) with a novel nanoscale contrast agent to examine tumor microenvironment characteristics noninvasively and in real-time. We demonstrate that CEUS imaging can: (1) evaluate tumor microenvironment features and (2) be used to help predict the distribution of doxorubicin-loaded liposomes in the tumor parenchyma. CEUS using nanobubbles (NBs) was carried out in two tumor types of high (LS174T) and low (U87) vascular permeability, and time-intensity curve (TIC) parameters were evaluated in both models prior to injection of doxorubicin liposomes. Consistently, LS174T tumors showed significantly different TIC parameters, including area under the rising curve (2.7x), time to peak intensity (1.9x) and decorrelation time (DT, 1.9x) compared to U87 tumors. Importantly, the DT parameter successfully predicted tumoral nanoparticle distribution (r = 0.86 {+/-} 0.13). Ultimately, substantial differences in NB-CEUS generated parameters between LS174T and U87 tumors suggest that this method may be useful in determining tumor vascular permeability and could be used as a biomarker for identifying tumor characteristics and predicting sensitivity to nanoparticle-based therapies. These findings could ultimately be applied to predicting treatment efficacy and to evaluating EPR in other diseases with pathologically permeable vasculature.

bioengineering↗

bioRxiv2022

Decorrelation Time Mapping as an Analysis Tool for Nanobubble-Based Contrast Enhanced Ultrasound Imaging

Nanobubbles (NBs) are nanoscale ([~]100-500 nm diameter) ultrasound (US) contrast agents that enable new robust applications of contrast enhanced US and US-mediated therapy. Due to their sub-micron size, high particle density, and highly deformable shell, NBs exhibit unique properties. In pathological states of heightened vascular permeability, such as in tumours, NBs can extravasate, enabling extravascular applications not currently possible with clinically available microbubbles ([~]1000-10,000 nm diameter). This ability can be explored to develop imaging biomarkers to improve tumour detection. There is a need for an imaging method that can rapidly and effectively separate intravascular versus extravascular NB signal when imaged using nonlinear dynamic contrast enhanced US. Herein, we demonstrated the use of decorrelation time (DT) mapping to achieve this goal. Two in vitro models were used to explore the roles of NB velocity and diffusion on DTs. Mice bearing prostate specific membrane antigen (PSMA) expressing flank tumours (n = 7) were injected with bubble agents to evaluate the in vivo potential of this technique. The DT was calculated at each pixel of nonlinear contrast videos to produce DT maps. Across all models, long DT correlated with slowly moving or entrapped NBs while short DT correlated with flowing NBs. DT maps were sensitive to NBs in tumour tissue with high average DT in tumour regions ([~]10 s) compared to surrounding normal tissue ([~]1 s). Molecular NB targeting to PSMA extended DT (17 s) compared to non-targeted NBs (12 s), demonstrating sensitivity to NB adherence dynamics. Overall, DT mapping of in vivo NB dynamics produced detailed information of tumour tissue and showed potential for quantifying extravascular NB kinetics. This new NB-contrast enhanced US-based biomarker can be useful in molecular ultrasound imaging, with improved sensitivity and specificity of target tissue detection and potential for use as a predictor of vascular permeability and the enhanced permeability and retention (EPR) effect in tumours.

bioengineering↗

bioRxiv2022

High-frequency quantitative ultrasound to assess the acoustic properties of engineeredtissues in vitro

Acoustic properties of biomaterials and engineered tissues reflect their structure and cellularity. High-frequency ultrasound (US) can non-invasively characterize and monitor these properties with sub-millimetre resolution. We present an approach to estimate the acoustic properties of cell-laden hydrogels that accounts for frequency-dependent effects of attenuation in coupling media, hydrogel thickness, and interfacial transmission/reflection coefficients of US waves, all of which can bias attenuation estimates. Cell-seeded fibrin hydrogel disks were raster-scanned using a 40 MHz US transducer. Thickness, speed of sound, acoustic impedance, and acoustic attenuation coefficients were determined from the difference in the time-of-flight and ratios of the magnitudes of US signals, interfacial transmission/reflection coefficients, and acoustic properties of the coupling media. With this approach, hydrogel thickness was accurately measured by US, with excellent agreement to confocal microscopy (r2 = 0.97). Accurate thickness measurement enabled acoustic property measurements that were independent of hydrogel thickness, despite up to 60% reduction in thickness due to cell-mediated contraction. Notably, acoustic attenuation coefficients increased with increasing cell concentration (p<0.001), reflecting hydrogel cellularity independent of contracted hydrogel thickness. This approach enables accurate measurement of the intrinsic acoustic properties of biomaterials and engineered tissues to provide new insights into their structure and cellularity.

bioengineering↗

bioRxiv2022

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 gradually increased by 22.8 {+/-} 13.1% throughout our experiment, with peak intensity reached within 145 seconds. In contrast, nanobubbles in PBS had a stable signal with negligible change in intensity (-1.7 {+/-} 3.2%) over 8 minutes. Under the same conditions, microbubbles made with the same lipid formulation showed a -56.8 {+/-} 6.1% decrease in enhancement. 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.

bioengineering↗

bioRxiv2018

Label-free assessment of red blood cell storage lesions by deep learning

Blood transfusion is a life-saving clinical procedure. With millions of units needed globally each year, it is a growing concern to improve product quality and recipient outcomes.\n\nStored red blood cells (RBCs) undergo continuous degradation, leading to structural and biochemical changes. To analyze RBC storage lesions, complex biochemical and biophysical assays are often employed.\n\nWe demonstrate that label-free imaging flow cytometry and deep learning can characterize RBC morphologies during 42-day storage, replacing the current practice of manually quantifying a blood smear from stored blood units. Based only on bright field and dark field images, our model achieved 90% accuracy in classifying six different RBC morphologies associated with storage lesions versus human-curated manual examination. A model fitted to the deep learning-extracted features revealed a pattern of morphological changes within the aging blood unit that allowed predicting the expiration date of stored blood using solely morphological assessment.\n\nDeep learning and label-free imaging flow cytometry could therefore be applied to reduce complex laboratory procedures and facilitate robust and objective characterization of blood samples.

bioinformatics↗