Echographic and physical characterization of albumin-stabilized nanobubbles.

There has been increasing interest in using nanobubbles (NBs) for ultrasound mediated drug delivery as well as for ultrasound imaging. Albumin NBs are especially attractive for its potential of becoming a versatile platform for drug carriers and molecular targeted therapy agents. However, physical characterization of NBs is generally considered to be difficult due to various technical issues, such as concentration limitations, nanoparticle contamination, etc. In the present study, we measured the size distribution, concentration and weight density of albumin stabilized NBs by means of multiple nanoscale measurement modalities. Laser nanoparticle tracking analysis, multicolor flow cytometry, resonance mass evaluation showed consistent measurement results of the NBs with low mass weight density and diameter size ranging from 100 nm to 400 nm. Furthermore, the NB solution showed excellent images by high frequency ultrasound (30-50 MHz) in flow model acoustic phantoms. The NBs also induced acute cell disruption by low intensity ultrasound (0.8 W/cm2) irradiation. We successfully fabricated and characterized albumin stabilized NBs which could serve as an effective platform for future theranositic agents.

Enhanced cell killing and apoptosis of oral squamous cell carcinoma cells with ultrasound in combination with cetuximab coated albumin microbubbles.

Targeted microbubbles have the potential to be used for ultrasound (US) therapy and diagnosis of various cancers. In the present study, US was irradiated to oral squamous cell carcinoma cells (HSC-2) in the presence of cetuximab-coated albumin microbubbles (CCAM). Cell killing rate with US treatment at 0.9 W/cm2 and 1.0 W/cm2 in the presence of CCAM was greater compared to non-targeted albumin microbubbles (p < .05). On the other hand, selective cell killing was not observed in human myelomonocytic lymphoma cell line (U937) that had no affinity to cetuximab. Furthermore, US irradiation in the presence of CCAM showed a fivefold increase of cell apoptotic rate for HSC-2 cells (21.0 ± 3.8%) as compared to U937 cells (4.0 ± 0.8%). Time-signal intensity curve in a tissue phantom demonstrated clear visualisation of CCAM with conventional US imaging device. Our experiment verifies the hypothesis that CCAM was selective to HSC-2 cells and may be applied as a novel therapeutic/diagnostic microbubble for oral squamous cell carcinoma.

Optical observation of cell sonoporation with low intensity ultrasound.

Sonoporation is a promising drug delivery technique with great potential in medicine. However, its applications have been limited mostly by the lack of understanding its underlying biophysical mechanism, partly due to the inadequacy of the existing models for coupling with highly sensitive imaging techniques to directly observe the actual precursor events of cell-microbubble interaction under low intensity ultrasound. Here, we introduce a new in vitro method utilizing capillary-microgripping system and micro-transducer to achieve maximum level of experimental flexibility for capturing real time highly magnified images of cell-microbubble interaction, hitherto unseen in this context. Insonation of isolated single cells and microbubbles parallel with high speed microphotography and fluorescence microscopy allowed us to identify dynamic responses of cell-membrane/microbubble in correlation with sonoporation. Our results showed that bubble motion and linear oscillation in close contact with the cell membrane can cause local deformation and transient porosity in the cell membrane without rupturing it. This method can also be used as an in situ gene/drug delivery system of targeted cells for non-invasive clinical applications.

Thermal dissection of lentil seedling amine oxidase domains by differential scanning calorimetry.

The relationships between the structural and energetic domains of lentil seedling amine oxidase (LSAO) were investigated using modifiers that target the active site and the carbohydrate moiety of the enzyme. An irreversible inhibitor, aminoguanidine, specifically modified the active site of the lentil enzyme, whereas sodium metaperiodate cleaves carbohydrate moieties covalently bound to the native enzyme. Differential scanning calorimetry (DSC) measurements were made on the modified LSAOs. Deconvolution of the reversible thermal DSC profiles of the modified enzyme gave three subpeaks (energetic domains), each of which was assigned to one of the three structural domains of the native protein. Our results led us to conclude that deglycosylation of LSAO has no effect on thermal stability, whereas binding of the inhibitor imparts more stability to the enzyme.