Fig. 4: Cytosolic delivery of large macromolecules by laser-activated NBs in adherent HeLa cells. | Nature Communications

Fig. 4: Cytosolic delivery of large macromolecules by laser-activated NBs in adherent HeLa cells.

From: Light triggered nanoscale biolistics for efficient intracellular delivery of functional macromolecules in mammalian cells

Fig. 4

a Schematic overview of the direct cytoplasmic delivery mechanism by laser-activated NBs: (i) NBs are co-incubated for a certain period of time with the macromolecules of interest (e.g., FITC-dextran) followed by exposure to one single laser pulse at the VB threshold; (ii) upon laser activation, the nanoprojectiles will puncture the cell membrane of nearby cells, generating transient pores through which the exogenous compounds can enter directly to the cell’s cytosol; (iii) following membrane repair, cells are loaded with the macromolecules. b Optimization of the delivery efficiency of FD500 (2 mg/mL) in HeLa cells by 200-NBs with polystyrene beads as nanoprojectiles. Cell viability was determined for all experiments by a CellTiter-Glo assay post-delivery. 200-NBs were activated in every case by irradiation at the VB threshold fluence of 1.22 J/cm2 (7 ns pulse at λ = 561 nm), unless otherwise specified. Controls were always included of cells incubated with FD500 and a mixture of uncoupled IONP cores at the same concentration as the NBs and polystyrene beads at a 1% v/v excess. (i) Effect of the concentration of 200-NBs when immediately irradiated upon addition to the cell medium together with FD500 (’0 min incubation’); (ii) effect of the nanoprojectile size of 100-NBs, 200-NBs, and 500-NBs and direct irradiation (’0 min incubation’) using a fixed concentration of 1.3 × 108 NBs/mL; (iii) effect of the incubation time of 200-NBs using a concentration of 1.3 × 108 NBs/mL. The vertical orange dashed line indicates the condition selected for further experiments. (iv) Comparison of delivery efficiency and cell viability with different irradiation set-ups. Irradiation was performed at the respective VB threshold fluences. c Delivery efficiency of molecular probes of different sizes using 200-NBs: FD10, FD150 and FD500. Effective concentration = 2 mg/mL. d Effect of the nanoprojectiles density on the delivery of FD500 using 200-NBs: polystyrene (1.04 g/cm3); PLGA (1.3 g/cm3); Titania (4.23 g/cm3). The vertical orange dashed line highlights results obtained with PLGA NPs as nanoprojectiles, which would be used in further transfection experiments. All results presented correspond to mean ± SD of n = 3 biologically independent samples.

Back to article page