Efficient Intracellular Delivery of Molecules with High Cell Viability Using Nanosecond-Pulsed Laser-Activated Carbon Nanoparticles

Abstract

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.

Laser-assisted photoporation: fundamentals, technological advances and applications

“…Carbon black (CB) NP-sensitized photoporation was first reported by the group of Prausnitz [77,78]. They reported that thermal effects are not the main reason for membrane poration.…”

“…In a comparative study of thermal vs. VNB mediated membrane poration, it has been found that VNB-mediated membrane poration actually allows more efficient uptake of exogenous nanomaterials with reduced cytotoxicity as compared to thermal poration at low laser intensity [76]. Although AuNPs are the most widely used for membrane poration, other nanomaterials like titanium nanostructures and carbon-based nanomaterials are also currently being evaluated as alternative sensitizing NPs [77][78][79][80].…”

Delivery of siRNA to Ovarian Cancer Cells Using Laser-Activated Carbon Nanoparticles

“…In this method, we expose CB nanoparticles to nanosecond pulsed laser, causing the nanoparticles to preferentially heat up, which results in particle expansion [27], liquid vaporization [27,28] and/or chemical reaction (C[s] + H 2 O[l] → CO[g] + H 2 [g]) [29], followed by generation of acoustic waves, leading to poration of cell membranes [30,31]. Molecules then passively transport into the cell without the need of endocytosis.…”

“…Molecules then passively transport into the cell without the need of endocytosis. Previously we demonstrated this method to be efficient in delivering small molecules like calcein and larger proteins like bovine serum albumin [30,31]; here we seek to extend the application to siRNA not only to show intracellular uptake of siRNA, but to demonstrate RNA interference by knocking down expression of a specific gene by delivery of biologically active siRNA.…”

“…In our approach, the nanoparticles transduce laser energy (i.e., photons) into mechanical energy (i.e., acoustic waves) that impact the cell membrane to increase its permeability [31]. In this way, siRNA provided in the extracellular medium can then diffuse directly into its area of target, in other words, cytoplasm, where the mature mRNAs are present.…”