Abstract
Recently, we used a recombinant produced C-terminus (D194-F319) of the Clostridium perfringens enterotoxin (C-CPE) to functionalize gold nanoparticles (AuNPs) for a subsequent specific killing of claudin expressing tumor cells using the gold nanoparticle-mediated laser perforation (GNOME-LP) technique. For a future in vivo application, it will be crucial to know the physical parameters and the biological mechanisms inducing cell death for a rational adaptation of the system to real time situation. Regarding the AuNP functionalization, we observed that a relationship of 2.5 × 10−11 AuNP/mL to 20 µg/mL C-CPE maximized the killing efficiency. Regardingphysical parameters, a laser fluence up to 30 mJ/cm2 increased the killing efficiency. Independent from the applied laser fluence, the maximal killing efficiency was achieved at a scanning velocity of 5 mm/s. In 3D matrigel culture system, the GNOME-LP/C-CPE-AuNP completely destroyed spheroids composed of Caco-2 cells and reduced OE-33 cell spheroid formation. At the biology level, GNOME-LP/C-CPE-AuNP-treated cells bound annexin V and showed reduced mitochondria activity. However, an increased caspase-3/7 activity in the cells was not found. Similarly, DNA analysis revealed no apoptosis-related DNA ladder. The results suggest that the GNOME-LP/C-CPE-AuNP treatment induced necrotic than apoptotic reaction in tumor cells.
Highlights
The gold nanoparticle-mediated (GNOME) laser perforation (GNOME-LP) technology was developed to achieve a gentle cell permeabilization allowing the entry of small molecule into cells while maintaining a maximal survival [1,2]
We showed that C-CPE -functionalized AuNPs in combination with GNOME-LP efficiently killed tumor cells expressing CLDN-3, -4, and -7, documented by the uptake of membrane impermeable of molecule such as propidium iodide [4]
We showed that a combination of GNOME-LP- and C-CPE-functionalized AuNPs could be used to kill claudin-expressing tumor cells [4]
Summary
The gold nanoparticle-mediated (GNOME) laser perforation (GNOME-LP) technology was developed to achieve a gentle cell permeabilization allowing the entry of small molecule into cells while maintaining a maximal survival [1,2]. In this context, the GNOME-LP technique was used for an accurate and high-throughput analysis of gap junction coupling [3] and for an efficient knock down of genes using specific siRNAs or morpholinos [2]. C-CPE-functionalized AuNPs (C-CPE-AuNPs) allowed specific targeting of cells and increased the killing efficiency [4]. The mechanisms of cell killing are still a matter of speculation, hindering a rational optimization of the technique in terms of maximal ablation of tumor cells with minimal action on non-tumor tissue
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