Delivery of molecules into cells using carbon nanoparticles activated by femtosecond laser pulses

Abstract

A major barrier to drug and gene delivery is crossing the cell's plasma membrane. Physical forces applied to cells via electroporation, ultrasound and laser irradiation generate nanoscale holes in the plasma membrane for direct delivery of drugs into the cytoplasm. Inspired by previous work showing that laser excitation of carbon nanoparticles can drive the carbon-steam reaction to generate highly controlled shock waves, we show that carbon black nanoparticles activated by femtosecond laser pulses can facilitate the delivery of small molecules, proteins and DNA into two types of cells. Our initial results suggest that interaction between the laser energy and carbon black nanoparticles may generate photoacoustic forces by chemical reaction to create transient holes in the membrane for intracellular delivery.

Figure 1

Intracellular uptake in cells exposed to femtosecond laser irradiation in presence of CB. Confocal micrographs show irradiated DU145 cells with uptake of calcein (a,b), FITC-BSA (c) and YOYO1-DNA (d). A large population of cells exhibited uptake of calcein when viewed at 10X magnification (a). Under 40X magnification, calcein (b) is seen at high concentration throughout the cell, including the nucleus (indicated by white arrow), while BSA (c) and DNA (d) were largely excluded from the nucleus. Samples were irradiated at 5mJ/cm² for 10 min in 30µg/ml CB. Scale bars are 100µm (a) and 5µm (b–d).

Figure 2

Effect of CB nanoparticle and cell type on uptake and viability. CB were used to deliver calcein and BSA proteins into DU145 cells (■) and GS-9L cells (). MWNT were used to deliver calcein into DU145 cells (). Graphs show percentage of cells with intracellular uptake (a), cell viability (b), and average number of molecules delivered per cell (c), as a function of nanoparticle and cell type. CB and MWNT were added at final concentrations of 30µg/ml. Samples were irradiated at 5mJ/cm² for 10 min. Data show average (n=3) ± SEM. *p<0.05 for cells with uptake of model drug compared to non-irradiated control (data not shown), #p<0.05 for cell viability compared to non-irradiated control, +p>0.05 for cell viability compared to non-irradiated control, ^p<0.05 for calcein molecule/cell compared to non-irradiated control, ^ap>0.05 for DU145 cells compared to GS-9L cells, ^bp<0.05 for DU145 cells compared to GS-9L, ^cp<0.05 for CB compared to MWNT. See S.I. Section 1.6 for statistical methods.

Figure 3

Uptake and expression of plasmid DNA. The graph shows intracellular uptake (■) and transfection () of luciferase plasmid DNA in DU145 cells. Uptake of YOYO1-labeled plasmid DNA was assayed < 2h after irradiation to assess intracellular delivery of DNA molecules. Luciferase expression was measured 48h after irradiation to assess expression of the luciferase protein encoded in the DNA. Each sample had 5×10⁵ cells and 30µg/ml CB. Irradiation was carried out at 5mJ/cm² for 10 min. Non-irradiated controls were identical to irradiated samples, except no laser irradiation was applied. Data show average (n=3) ± SEM. *p<0.05 for treated cells compared to non-irradiated negative control.

Figure 4

Effect of laser fluence and exposure time on intracellular calcein uptake and cell viability in DU145 cells. Samples were irradiated in 30µg/ml CB. Data show average (n=3) ± SEM. *p<0.05 for cells with uptake compared to non-irradiated control at the same exposure time, ⁺p<0.05 for cell viability compared to non-irradiated control at the same exposure time.

Figure 5

Effect of irradiation conditions on intracellular uptake of calcein in DU145 cells. Standard conditions for this experiment were irradiation at 5mJ/cm²for 10 min with 30µg/ml CB and 10µM calcein. Deviations from standard conditions are as follows. (A) Positive control (standard conditions). (B) Non-irradiated negative control (standard conditions without irradiation). (C) Irradiation without CB. (D) Irradiation with gold nanorods instead of CB. (E) Cells added <1s after irradiation of cell-free solution containing CB and calcein. (F) Irradiation in media with five-fold higher viscosity. (G) Irradiation of cells pre-treated in K⁺-depleted media to block endocytic processes. (H) Calcein added < 1s (H1), 30s (H2), 60s (H3) and 120s (H4) after irradiation for 3 min. Data are expressed as the percentage of cells with calcein uptake among all irradiated cells, except for (G), in which data are expressed as the percentage of viable cells with calcein. This correction was made in (G) because the K⁺-depletion pre-treatment to suppress endocytosis killed ~20% of cells. Data show average (n=3) ± SEM. *p<0.05 for cells with uptake compared to condition A, ⁺p>0.05 for cells with uptake compared to condition B, ^p>0.05 for cells with uptake compared to condition A.