Enhancing the therapeutic efficacy of doxorubicin-coated gold nanoparticles by laser irradiation

Conventional anticancer drugs have several limitations, including low solubility and selectivity. Nanotechnologies have emerged as advanced drug delivery systems (DDS) aimed at improving drug delivery precision and mitigating adverse effects. Among these, gold nanoparticles (GNPs) have gained attention for their unique properties, including surface chemistry, loading capacity, biocompatibility, and optical absorption. In this project, the increased therapeutic efficacy of gold nanoparticles following laser irradiation was analysed. GNPs were synthesised using the Turkevich method. Their surface was functionalised with an anticancer drug, doxorubicin, and a targeting agent, folate. Additionally, for particle tracking, a fluorescent molecule, Bodipy, was added. Stealth properties were crucial for reaching the target, so the surface was saturated with methoxy-polyethylene glycol-thiol. In vitro experiments were conducted at the University of Ghent; therefore, it was essential for the particles to remain stable during storage and transportation. This stability was achieved via a freeze-drying process. Polyvinylpyrrolidone and Tween 20 were chosen as cryoprotectants, and the stability of the samples was confirmed through dynamic light scattering (DLS) measurements and UV-Vis spectroscopy. The cytotoxicity of GNPs, combined with laser-induced photoporation, was evaluated in two cell lines: SKOV3 cells expressing folate receptors and A549 cells lacking these receptors. It was proven that laser energy enhanced doxorubicin cytotoxicity in a concentration and laser fluence-dependent manner, reducing the time required for therapeutic activity. Several methods were employed to understand the underlying mechanism, including investigating the generation of vapour nanobubbles (VNB). VNBs were generated only in cells treated with particles exhibiting cytotoxicity, probably due to high accumulation or cluster formation, which increases the photothermal behaviour. This characteristic of gold nanoparticles also allows the endosomal escape process to occur, as confirmed through confocal microscopy analysis combined with the use of Calcein. This process allows a greater release of doxorubicin into the cytosol, thereby increasing its cytotoxic effect. Transmission electron microscopy analysed the propensity for clustering by the particles, confirming a greater tendency in the case of folate coating. Moreover, flow cytometry was used to confirm the presence of particles inside cells. In summary, in vitro experiments provide compelling evidence of photoporation enhancing the cytotoxicity of doxorubicin-coated gold nanoparticles. This drug delivery system reduces doxorubicin's side effects and, when combined with laser energy, allows the administration of lower DDS concentrations and accelerates the onset of therapeutic activity.