Optical and electron microscopy study of laser-based intracellular molecule delivery using peptide-conjugated photodispersible gold nanoparticle agglomerates.

BACKGROUND: Cell-penetrating peptides (CPPs) can act as carriers for therapeutic molecules such as drugs and genetic constructs for medical applications. The triggered release of the molecule into the cytoplasm can be crucial to its effective delivery. Hence, we implemented and characterized laser interaction with defined gold nanoparticle agglomerates conjugated to CPPs which enables efficient endosomal rupture and intracellular release of molecules transported. RESULTS: Gold nanoparticles generated by pulsed laser ablation in liquid were conjugated with CPPs forming agglomerates and the intracellular release of molecules was triggered via pulsed laser irradiation (γ = 532 nm, τ pulse = 1 ns). The CPPs enhance the uptake of the agglomerates along with the cargo which can be co-incubated with the agglomerates. The interaction of incident laser light with gold nanoparticle agglomerates leads to heat deposition and field enhancement in the vicinity of the particles. This highly precise effect deagglomerates the nanoparticles and disrupts the enclosing endosomal membrane. Transmission electron microscopy images confirmed this rupture for radiant exposures of 25 mJ/cm2 and above. Successful intracellular release was shown using the fluorescent dye calcein. For a radiant exposure of 35 mJ/cm2 we found calcein delivery in 81 % of the treated cells while maintaining a high percentage of cell viability. Furthermore, cell proliferation and metabolic activity were not reduced 72 h after the treatment. CONCLUSION: CPPs trigger the uptake of the gold nanoparticle agglomerates via endocytosis and co-resident molecules in the endosomes are released by applying laser irradiation, preventing their intraendosomal degradation. Due to the highly localized effect, the cell membrane integrity is not affected. Therefore, this technique can be an efficient tool for spatially and temporally confined intracellular release. The utilization of specifically designed photodispersible gold nanoparticle agglomerates (65 nm) can open novel avenues in imaging and molecule delivery. Due to the induced deagglomeration the primary, small particles (~5 nm) are more likely to be removed from the body.

Structural damage of Bacillus subtilis biofilms using pulsed laser interaction with gold thin films.

There is a huge interest in developing strategies to effectively eliminate biofilms due to their negative impact in both industrial and clinical settings. In this study, structural damage was induced on two day-old B. subtilis biofilms using the interaction of 532 nm pulsed laser with gold thin films. Radiant exposure of 225 mJ/cm2 induced distinct changes on the surface structure and overall morphology of the matured biofilms after laser irradiation. Moreover, at the radiant exposure used, changes in the colour and viscosity of the biofilm were observed which may indicate a compromised extracellular matrix. Irradiated biofilms in the presence of gold film also showed strong propidium iodide signal which implies an increase in the number of dead bacterial cells after laser treatment. Thus, this laser-based technique is a promising approach in targeting and eradicating matured biofilms attached on surfaces such as medical implants.

Gold Nanoparticle-Mediated Delivery of Molecules into Primary Human Gingival Fibroblasts Using ns-Laser Pulses: A Pilot Study.

Interaction of gold nanoparticles (AuNPs) in the vicinity of cells' membrane with a pulsed laser (λ = 532 nm, τ = 1 ns) leads to perforation of the cell membrane, thereby allowing extracellular molecules to diffuse into the cell. The objective of this study was to develop an experimental setting to deliver molecules into primary human gingival fibroblasts (pHFIB-G) by using ns-laser pulses interacting with AuNPs (study group). To compare the parameters required for manipulation of pHFIB-G with those needed for cell lines, a canine pleomorphic adenoma cell line (ZMTH3) was used (control group). Non-laser-treated cells incubated with AuNPs and the delivery molecules served as negative control. Laser irradiation (up to 35 mJ/cm²) resulted in a significant proportion of manipulated fibroblasts (up to 85%, compared to non-irradiated cells: p < 0.05), while cell viability (97%) was not reduced significantly. pHFIB-G were perforated as efficiently as ZMTH3. No significant decrease of metabolic cell activity was observed up to 72 h after laser treatment. The fibroblasts took up dextrans with molecular weights up to 500 kDa. Interaction of AuNPs and a pulsed laser beam yields a spatially selective technique for manipulation of even primary cells such as pHFIB-G in high throughput.