Conventional physical and chemical methods that efficiently deliver molecules into cells are often associated with low cell viability. In this study, we evaluated the cellular effects of carbon nanoparticles believed to emit photoacoustic waves due to nanosecond-pulse laser activation to test the hypothesis that this method could achieve efficient intracellular delivery while maintaining high cell viability. Suspensions of DU145 human prostate carcinoma cells, carbon black (CB) nanoparticles, and calcein were exposed to 5–9 ns long laser pulses of near-infrared (1064 nm wavelength) light and then analyzed by flow cytometry for intracellular uptake of calcein and cell viability by propidium iodide staining. We found that intracellular uptake increased and in some cases saturated at high levels with only small losses in cell viability as a result of increasing laser fluence, laser exposure time, and as a unifying parameter, the total laser energy. Changing interpulse spacing between 0.1 and 10 s intervals showed no significant change in bioeffects, suggesting that the effects of each pulse were independent when spaced by at least 0.1 s intervals. Pretreatment of CB nanoparticles to intense laser exposure followed by mixing with cells also had no significant effect on uptake or viability. Similar uptake and viability were seen when CB nanoparticles were substituted with India ink, when DU145 cells were substituted with H9c2 rat cardiomyoblast cells, and when calcein was substituted with FITC-dextran. The best laser exposure conditions tested led to 88% of cells with intracellular uptake and close to 100% viability, indicating that nanosecond-pulse laser-activated carbon nanoparticles can achieve efficient intracellular delivery while maintaining high cell viability.

Efficient intracellular delivery of molecules is needed to modulate cellular behavior for laboratory and medical applications, but is often limited by trade-offs between achieving high intracellular delivery and maintaining high cell viability. Here, we studied photoacoustic delivery of molecules into cells by exposing DU145 human prostate carcinoma cells to nanosecond laser pulses in the presence of carbon black nanoparticles. Under strong laser exposure conditions, less than 30% of cells were viable and exhibited uptake. Addition of poloxamer surfactant at those laser exposure conditions increased cell viability to almost 90%, with intracellular uptake in >80% of cells. This remarkable increase in efficiency of intracellular delivery and cell viability may be attributed to enhanced cell membrane resealing by poloxamer surfactant after photoacoustic delivery. While F-68 poloxamer was effective, the larger, more-hydrophobic F-127 poloxamer provided the best results. There was no significant protective effect from addition of Ca(2+) , BAPTA-AM, ATP, fetal bovine serum or glycine betaine, which were expected to promote active cell membrane repair mechanisms and other active intracellular protective processes. We conclude that poloxamer surfactant preserves cell viability during photoacoustic delivery of molecules into cells, thereby enabling highly efficient intracellular delivery.

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