Laser irradiated fluorescent perfluorocarbon microparticles in 2-D and 3-D breast cancer cell models

Abstract

Perfluorocarbon (PFC) droplets were studied as new generation ultrasound contrast agents via acoustic or optical droplet vaporization (ADV or ODV). Little is known about the ODV irradiated vaporization mechanisms of PFC-microparticle complexs and the stability of the new bubbles produced. In this study, fluorescent perfluorohexane (PFH) poly(lactic-co-glycolic acid) (PLGA) particles were used as a model to study the process of particle vaporization and bubble stability following excitation in two-dimensional (2-D) and three-dimensional (3-D) cell models. We observed localization of the fluorescent agent on the microparticle coating material initially and after vaporization under fluorescence microscopy. Furthermore, the stability and growth dynamics of the newly created bubbles were observed for 11 min following vaporization. The particles were co-cultured with 2-D cells to form 3-D spheroids and could be vaporized even when encapsulated within the spheroids via laser irradiation, which provides an effective basis for further work.

Figure 1. SEM, fluorescent microscopy, and confocal microscopy images of a DiI-labelled microparticle.

(A) SEM image; (B) Fluorescent microscopy image; (C) Confocal microscopy images of a DiI-labelled microparticle from the surface plane to the inner planes. Scale bars represent 5 μm.

Figure 2. Brightfield and fluorescent microscopy images of a vaporized DiI-labelled bubble.

(A) Brightfield image; (B) Fluorescent microscopy image; (C) Merged picture of (A,B). Scale bars represent 10 μm.

Figure 3. SEM images of a DiI-labelled microparticle before and after vaporization.

(A) Before vaporization; (B) After vaporization. Arrows points to the area of the microparticle where vaporization occurred under the influence of the high electron beam.

Figure 4. The sequence shows a 7.5 μm PFC liquid microparticle expanding and the newly bubble emerging over time upon laser irradiation, over the first 630 s.

Scale bars represent 20 μm.

Figure 5. Brightfield and fluorescent images of a fluorescent microparticle before vaporization and the newly created bubble after vaporization.

(A) Brightfield image of a microparticle before vaporization; (B) Brightfield image of the newly created bubble, which was taken approximately 11 s after vaporization; (C) Fluorescent image of a microparticle before vaporization; (D) Fluorescent image taken approximately 20 s after the brightfield image, which shows that after vaporization the fluorescence signal is localized to the interface between the new bubble and the original microparticle, with no apparent fluorescence visible within the newly created bubble. Scale bars represent 20 μm.

Figure 6. Increase in bubble diameter relative to the initial microparticle size as a function of time after vaporization in the first 2 s and 11 min after vaporization.

(A) Bubble size change over the first 2 s; (B) Bubble size change over the first 11 min. The lines represent the mean ± standard deviation measurements of bubble size change, where 5 microparticles were monitored.

Figure 7. Fluorescent microscopy images of the MCF-7 cellular uptake of DiI-labelled microparticles and their intracellular distribution.

(A) Under 549 nm laser excitation, red fluorescence represents microparticles; (B) Under 488 nm laser excitation, green fluorescence represents cells; (C) Merged picture of (A,B); (D) Brightfield image. After incubation with DiI-labelled microparticles for 4 h, many microparticles had been phagocytized by the cells. Scale bars represent 20 μm.

Figure 8. Fluorescent and brightfield micrographs of laser-vaporized DiI-labelled microparticles encapsulated in MCF-7 cells.

(A) Under 549 nm laser excitation, red fluorescence represents microparticles; (B) Under 488 nm laser excitation, green fluorescence represents cells; (C) Merged picture of (A,B). (D) Brightfield image. Scale bars represent 20 μm.

Figure 9. Confocal micrographs of the MCF-7 spheroid uptake of DiI-labelled 3-D microparticles and their intracellular distribution.

The green fluorescence on the left represents cells under 488 nm laser excitation, the red fluorescence in the middle represents microparticles under 549 nm laser excitation; the green and red fluorescence on the right depicts the merged images of both the red and green fluorescence. After incubation with DiI-labelled microparticles for 7 days, 2-D tumor cells successfully formed 3-D spheroids. The green fluorescence labelled the cytoplasm and the red fluorescence labelled the microparticles, which accumulated in the cytoplasm. The distribution of the microparticles was analyzed by confocal microscopy using Z-stack imaging with 300 μm intervals. Scale bars represent 100 μm.

Figure 10. Fluorescent and brightfield micrographs of MCF-7 spheroid-encapsulated, laser-vaporized DiI-labelled microparticles.

(A) Under 549 nm laser excitation, red fluorescence represents microparticles; (B) Under 488 nm laser excitation, green fluorescence represents cells; (C) Merged image of (A,B). (D) Brightfield image. Scale bars represent 50 μm.

Figure 11. Cells viabilities of DiI-labelled microparticles on breast cancer cell viability with or without 532-nm laser irradiation.

Figure 12

(A) Experimental setup used for optical observation of microparticle vaporization. The laser was focused through the platform onto the sample. Wavelengths of 500–650 nm were reflected by the mirror toward the sample, but other wavelengths were also allowed to pass for optical viewing. (B) Schematic depicting the vaporization of a single microparticle under laser irradiation.