RESUMEN
How nanoparticles distribute in living cells and overcome cellular barriers are important criteria in the design of drug carriers. Pair-correlation microscopy is a correlation analysis of fluctuation in the fluorescence intensity obtained by a confocal line scan that can quantify the dynamic properties of nanoparticle diffusion including the number of mobile nanoparticles, diffusion coefficient, and transit time across a spatial distance. Due to the potential heterogeneities in nanoparticle properties and the complexity within the cellular environment, quantification of averaged auto- and pair-correlation profiles may obscure important insights into the ability of nanoparticles to deliver drugs. To overcome this issue, we used phasor analysis to develop a data standardizing method, which can segment the scanned line into several subregions according to diffusion and address the spatial heterogeneity of nanoparticles moving inside cells. The phasor analysis is a fit-free method that represents autocorrelation profiles for each pixel relative to free diffusion on the so-called phasor plots. Phasor plots can then be used to select subpopulations for which the auto- and pair-correlation analysis can be performed separately. We demonstrate the phasor analysis for pair-correlation microscopy for investigating 16 nm, Cy5-labeled silica nanoparticles diffusing across the plasma membrane and green fluorescent proteins (GFP) diffusing across nuclear envelope in MCF-7 cells.
Asunto(s)
Nanopartículas , Difusión , Portadores de Fármacos , Humanos , Microscopía Confocal , Microscopía Fluorescente , Dióxido de SilicioRESUMEN
An effective strategy for regioselective modification and directional assembly of anisotropic nanoparticles is demonstrated to explore the electric field enhancement in assembled gold nanobipyramids compared with gold nanorods. The well-defined secondary plasmonic hot spots between the coupled gold nanobipyramids exhibit the capability for single molecule detection.
RESUMEN
Sonoporation has been associated with drug delivery across cell membranes and into target cells, yet several limitations have prohibited further advancement of this technology. Higher delivery rates were associated with increased cellular death, thus implying a safety-efficacy trade-off. Meanwhile, there has been no reported study of safe in vitro sonoporation in a physiologically relevant flow environment. The objective of our study was not only to evaluate sonoporation under physiologically relevant flow conditions, such as fluid velocity, shear stress and temperature, but also to design ultrasound parameters that exploit the presence of flow to maximize sonoporation efficacy while minimizing or avoiding cellular damage. Human umbilical vein endothelial cells (EA.hy926) were seeded in flow chambers as a monolayer to mimic the endothelium. A peristaltic pump maintained a constant fluid velocity of 12.5 cm/s. A focused 0.5 MHz transducer was used to sonicate the cells, while an inserted focused 7.5 MHz passive cavitation detector monitored microbubble-seeded cavitation emissions. Under these conditions, propidium iodide, which is normally impermeable to the cell membrane, was traced to determine whether it could enter cells after sonication. Meanwhile, calcein-AM was used as a cell viability marker. A range of focused ultrasound parameters was explored, with several unique bioeffects observed: cell detachment, preservation of cell viability with no membrane penetration, cell death and preservation of cell viability with sonoporation. The parameters were then modified further to produce safe sonoporation with minimal cell death. To increase the number of favourable cavitation events, we lowered the ultrasound exposure pressure to 40 kPapk-neg and increased the number of cavitation nuclei by 50 times to produce a trans-membrane delivery rate of 62.6% ± 4.3% with a cell viability of 95% ± 4.2%. Furthermore, acoustic cavitation analysis showed that the low pressure sonication produced stable and non-inertial cavitation throughout the pulse sequence. To our knowledge, this is the first study to demonstrate a high drug delivery rate coupled with high cell viability in a physiologically relevant in vitro flow system.
Asunto(s)
Membrana Celular/química , Células Endoteliales/química , Fluorocarburos/química , Fluorocarburos/efectos de la radiación , Lípidos/efectos de la radiación , Sonicación/métodos , Velocidad del Flujo Sanguíneo/fisiología , Línea Celular , Membrana Celular/efectos de la radiación , Electroporación/métodos , Células Endoteliales/fisiología , Células Endoteliales/efectos de la radiación , Humanos , Lípidos/química , MicroburbujasRESUMEN
Focused ultrasound and microbubbles have been extensively used to generate therapeutic bioeffects. Despite encouraging in vivo results, there remains poor control of the magnitude and spatial distribution of these bioeffects due to the limited ability of conventional pulse shapes and sequences to control cavitation dynamics. Thus current techniques are restricted by an efficacy-safety trade-off. The primary aim of the present study was to incorporate the presence of flow in the design of new short pulse sequences, which can more uniformly distribute the cavitation activity. Microbubbles flowing (fluid velocity: 10 mm s(-1)) through a 300 µm tube were sonicated with a focused 0.5 MHz transducer while acoustic emissions were captured with an inserted focused 7.5 MHz passive cavitation detector. The two foci were co-axially aligned and their focal points were overlapped. Whereas conventional sequences are composed of a long burst (>10,000 cycles) emitted at a low burst repetition frequency (<10 Hz), we decomposed this burst into short pulses by adding intervals to facilitate inter-pulse microbubble movement. To evaluate how this sequence influenced cavitation distribution, we emitted short pulses (peak-rarefactional pressure (PRP): 40-366 kPa, pulse length (PL): 5-25 cycles) at high pulse repetition frequencies (PRF: 0.625-10 kHz) for a burst length of 100 ms. Increased cavitation persistence, implied by the duration of the microbubble acoustic emissions, was a measure of improved distribution due to the presence of flow. Sonication at lower acoustic pressures, longer pulse intervals and lower PLs improved the spatial distribution of cavitation. Furthermore, spectral analysis of the microbubble emissions revealed that the improvement at low pressures is due to persisting stable cavitation. In conclusion, new short-pulse sequences were shown to improve spatiotemporal control of acoustic cavitation dynamics during physiologically relevant flow. This could lead to adjustable distribution of the generated in vivo bioeffect and therefore efficient and safe treatment of a wide range of pathologies.