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.
A critical issue is the biocompatibility of nanoparticle agglomerates. Here previous studies report on the advantages of biodegradable agglomerates cross-linked by polymers which dissipate in a cellular environment, while the resulting small nanoparticles can be easily cleared from the body via the kidney [41, 42]. The utilization of this concept in combination with laser-induced deagglomeration, however, has not been previously examined. It should be noted that small and ultrasmall gold nanoparticles, released after deagglomeration, have been previously reported to be cytotoxic [43, 44]. However, a recent comparison of toxicological studies has shown that these effects are predominantly caused by unrealistically high surface doses  which will never be reached in potential biomedical applications. We could, in addition, verify that small laser-generated nanoparticles, very similar to those applied in this study, are highly biocompatible, as they were proven not to interfere with critical functional cell parameters like oocyte maturation, even though the particles were taken up by the cells .
TEM-images of gold nanoparticles conjugated to CPP-AuNPs. CPP-AuNP-agglomerate in solution (a) and cells with CPP-AuNPs taken up via endocytosis (b, c). Endosomes with AuNPs can be found in different stages of maturation (b), EE early endosome, LE late endosome, L lysosome). c shows a more general overview of a cell containing endosomes with CPP-AuNPs. Scale bars: a 100 nm, b 500 nm, inset 100 nm, c 800 nm
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