Photothermal induction of pyroptosis in malignant glioma spheroids using (16-mercaptohexadecyl)trimethylammonium bromide-modified cationic gold nanorods.

Plasmonic photothermal therapy (PPTT) employing plasmonic gold nanorods (GNRs) presents a potent strategy for eradication of tumors including aggressive brain gliomas. Despite its promise, there is a pressing need for a more comprehensive evaluation of PPTT using sophisticated in vitro models that closely resemble tumor tissues, thereby facilitating the elucidation of therapeutic mechanisms. In this study, we exposed 3D glioma spheroids (tumoroids) to (16-mercaptohexadecyl)trimethylammonium bromide-functionalized gold nanorods (MTAB-GNRs) and a near-infrared (NIR) laser. We demonstrate that the photothermal effect can be fine-tuned by adjusting the nanoparticle concentration and laser power. Depending on the selected parameters, the laser can trigger either regulated or non-regulated cell death (necrosis) in both mouse GL261 and human U-87 MG glioma cell lines, accompanied by translocation of phosphatidylserine in the membrane. Our investigation into the mechanism of regulated cell death induced by PPTT revealed an absence of markers associated with classical apoptosis pathways, such as cleaved caspase 3. Instead, we observed the presence of cleaved caspase 1, gasdermin D, and elevated levels of NLRP3 in NIR-irradiated tumoroids, indicating the activation of pyroptosis. This finding correlates with previous observations of lysosomal accumulation of MTAB-GNRs and the known lysosomal pathway of pyroptosis activation. We further confirmed the absence of toxic breakdown products of GNRs using electron microscopy, which showed no melting or fragmentation of gold nanoparticles under the conditions causing regulated cell death. In conclusion, PPTT using coated gold nanorods offers significant potential for glioma cell elimination occurring through the activation of pyroptosis rather than classical apoptosis pathways.

Long-Term Accumulation, Biological Effects and Toxicity of BSA-Coated Gold Nanoparticles in the Mouse Liver, Spleen, and Kidneys.

Introduction: Gold nanoparticles are promising candidates as vehicles for drug delivery systems and could be developed into effective anticancer treatments. However, concerns about their safety need to be identified, addressed, and satisfactorily answered. Although gold nanoparticles are considered biocompatible and nontoxic, most of the toxicology evidence originates from in vitro studies, which may not reflect the responses in complex living organisms. Methods: We used an animal model to study the long-term effects of 20 nm spherical AuNPs coated with bovine serum albumin. Mice received a 1 mg/kg single intravenous dose of nanoparticles, and the biodistribution and accumulation, as well as the organ changes caused by the nanoparticles, were characterized in the liver, spleen, and kidneys during 120 days. Results: The amount of nanoparticles in the organs remained high at 120 days compared with day 1, showing a 39% reduction in the liver, a 53% increase in the spleen, and a 150% increase in the kidneys. The biological effects of chronic nanoparticle exposure were associated with early inflammatory and fibrotic responses in the organs and were more pronounced in the kidneys, despite a negligible amount of nanoparticles found in renal tissues. Conclusion: Our data suggest, that although AuNPs belong to the safest nanomaterial platforms nowadays, due to their slow tissue elimination leading to long-term accumulation in the biological systems, they may induce toxic responses in the vital organs, and so understanding of their long-term biological impact is important to consider their potential therapeutic applications.

The Effect of Chemical Structure of OEG Ligand Shells with Quaternary Ammonium Moiety on the Colloidal Stabilization, Cellular Uptake and Photothermal Stability of Gold Nanorods.

PURPOSE: Plasmonic photothermal cancer therapy by gold nanorods (GNRs) emerges as a promising tool for cancer treatment. The goal of this study was to design cationic oligoethylene glycol (OEG) compounds varying in hydrophobicity and molecular electrostatic potential as ligand shells of GNRs. Three series of ligands with different length of OEG chain (ethylene glycol units = 3, 4, 5) and variants of quaternary ammonium salts (QAS) as terminal functional group were synthesized and compared to a prototypical quaternary ammonium ligand with alkyl chain - (16-mercaptohexadecyl)trimethylammonium bromide (MTAB). METHODS: Step-by-step research approach starting with the preparation of compounds characterized by NMR and HRMS spectra, GNRs ligand exchange evaluation through characterization of cytotoxicity and GNRs cellular uptake was used. A method quantifying the reshaping of GNRs was applied to determine the effect of ligand structure on the heat transport from GNRs under fs-laser irradiation. RESULTS: Fourteen out of 18 synthesized OEG compounds successfully stabilized GNRs in the water. The colloidal stability of prepared GNRs in the cell culture medium decreased with the number of OEG units. In contrast, the cellular uptake of OEG+GNRs by HeLa cells increased with the length of OEG chain while the structure of the QAS group showed a minor role. Compared to MTAB, more hydrophilic OEG compounds exhibited nearly two order of magnitude lower cytotoxicity in free state and provided efficient cellular uptake of GNRs close to the level of MTAB. Regarding photothermal properties, OEG compounds evoked the photothermal reshaping of GNRs at lower peak fluence (14.8 mJ/cm2) of femtosecond laser irradiation than the alkanethiol MTAB. CONCLUSION: OEG+GNRs appear to be optimal for clinical applications with systemic administration of NPs not-requiring irradiation at high laser intensity such as drug delivery and photothermal therapy inducing apoptosis.

Highly hydrophilic cationic gold nanorods stabilized by novel quaternary ammonium surfactant with negligible cytotoxicity.

The photothermal cancer therapy using cationic gold nanorods (GNRs) stabilized by quaternary ammonium salts (QAS) have a great potential to enhance conventional cancer treatment as it promises the effective eradication of cancer cells including cells resistant to radio- and chemo-therapy and the stimulation of anti-tumor immune response. However, as the cytotoxicity of the conventional alkanethiol-QAS compounds limits their utility in medicine, here we developed GNRs modified by novel highly hydrophilic cationic surfactant composed of the quaternary ammonium group and ethylene glycol chain N,N,N-trimethyl-3,6,9,12,15-pentaoxaheptadecyl-17-sulfanyl-1-ammonium bromide (POSAB) showing insignificant cytotoxicity in the free state. Surface modification of GNRs by POSAB allowed to prepare nanoparticles with good stability in water, high cellular uptake and localization in lysosomes that are a promising alternative to alkanethiol-stabilized GNRs especially for biomedical applications.

Biological safety and tissue distribution of (16-mercaptohexadecyl)trimethylammonium bromide-modified cationic gold nanorods.

The exceptionally high cellular uptake of gold nanorods (GNRs) bearing cationic surfactants makes them a promising tool for biomedical applications. Given the known specific toxic and stress effects of some preparations of cationic nanoparticles, the purpose of this study was to evaluate, in an in vitro and in vivo in mouse, the potential harmful effects of GNRs coated with (16-mercaptohexadecyl)trimethylammonium bromide (MTABGNRs). Interestingly, even after cellular accumulation of high amounts of MTABGNRs sufficient for induction of photothermal effect, no genotoxicity (even after longer-term accumulation), induction of autophagy, destabilization of lysosomes (dominant organelles of their cellular destination), alterations of actin cytoskeleton, or in cell migration could be detected in vitro. In vivo, after intravenous administration, the majority of GNRs accumulated in mouse spleen followed by lungs and liver. Microscopic examination of the blood and spleen showed that GNRs interacted with white blood cells (mononuclear and polymorphonuclear leukocytes) and thrombocytes, and were delivered to the spleen red pulp mainly as GNR-thrombocyte complexes. Importantly, no acute toxic effects of MTABGNRs administered as 10 or 50 µg of gold per mice, as well as no pathological changes after their high accumulation in the spleen were observed, indicating good tolerance of MTABGNRs by living systems.

Two-Step Mechanism of Cellular Uptake of Cationic Gold Nanoparticles Modified by (16-Mercaptohexadecyl)trimethylammonium Bromide.

Cationic colloidal gold nanorods (GNRs) have a great potential as a theranostic tool for diverse medical applications. GNRs' properties such as cellular internalization and stability are determined by physicochemical characteristics of their surface coating. GNRs modified by (16-mercaptohexadecyl)trimethylammonium bromide (MTAB), MTABGNRs, show excellent cellular uptake. Despite their promise for biomedicine, however, relatively little is known about the cellular pathways that facilitate the uptake of GNRs, their subcellular fate and intracellular persistence. Here we studied the mechanism of cellular internalization and long-term fate of GNRs coated with MTAB, for which the synthesis was optimized to give higher yield, in various human cell types including normal diploid versus cancerous, and dividing versus nondividing (senescent) cells. The process of MTABGNRs internalization into their final destination in lysosomes proceeds in two steps: (1) fast passive adhesion to cell membrane mediated by sulfated proteoglycans occurring within minutes and (2) slower active transmembrane and intracellular transport of individual nanorods via clathrin-mediated endocytosis and of aggregated nanorods via macropinocytosis. The expression of sulfated proteoglycans was the major factor determining the extent of uptake by the respective cell types. Upon uptake into proliferating cells, MTABGNRs were diluted equally and relatively rapidly into daughter cells; however, in nondividing/senescent cells the loss of MTABGNRs was gradual and very modest, attributable mainly to exocytosis. Exocytosed MTABGNRs can again be internalized. These findings broaden our knowledge about cellular uptake of gold nanorods, a crucial prerequisite for future successful engineering of nanoparticles for biomedical applications such as photothermal cancer therapy or elimination of senescent cells as part of the emerging rejuvenation approach.