The development of targeting nanosystems for the treatment of glioblastoma and neuroblastoma tumours

Abstract

Chlorotoxin (CTX) peptide selectively targets glioblastoma multiforme (GB) and neuroblastoma (NB) and has excellent blood-brain barrier permeability. Therefore, CTX is a promising targeting molecule for nanoparticle (NP) based diagnostic and therapeutic applications. Bimetallic gold-platinum nanoparticles (AuPtNPs) have recently attracted great attention for anti-cancer and catalysis applications due to the synergistic effects of combined metal atoms which enhance NP properties when compared to their monometallic NP counterparts. Therefore, the aim of this study was to develop gold NPs (AuNPs) and AuPtNPs, both conjugated to CTX for treatment in GB and NB cancer cells in vitro. This was achieved by synthesizing two novel CTX-NPs through the preparation of citrate capped AuNPs and AuPtNPs which was modified using two different types of polyethylene glycol (PEG) molecules, which allowed for conjugation of CTX onto the NPs. The physicochemical properties of the NPs were characterized, and ultraviolet-visible absorbance spectroscopy analysis demonstrated an increase in the absorption maxima (λ max) of all AuNPs, while the absence of an λ max for all AuPtNPs confirmed the formation of bimetallic NPs. Dynamic light scattering analysis for both citrate capped NPs showed a hydrodynamic size of approximately 7 nm, which doubled after surface functionalization with PEG and slightly increased following CTX surface functionalization. The zeta-potential revealed highly stable citrate NPs and decreased to more neutral charges following PEG and CTX surface functionalization confirming surface modification of NPs which was further supported by fourier transform infra-red spectroscopy analysis. Transmission electron microscopy (TEM) measurements revealed roughly spherical and monodispersed NPs with a core size of approximately 5 nm for all NPs. NPs were stable in biological media over 48-hours and CTXNPs demonstrated binding and uptake to U87 human GB and SH-SY5Y human NB cancer cell lines. NP-induced toxicity was investigated using the WST-1 cell viability assay at concentrations of 75-300 µg/ml for 48 hours in U87 and SH-SY5Y cells, while the non-cancerous KMST-6 human fibroblast cells served as a control cell line. The maximal inhibitory concentrations (IC₅₀ values) for all NPs in U87 and SH-SY-5Y cells was generally similar, however the most promising results was revealed in U87 cells with CTX-AuPtNPs treatment and was further investigated and showed the induction of apoptosis (using the APOPercentage™ apoptosis assay), the production of reactive oxygen species (using the CM-H2DCFDA probe), and decrease in mitochondrial activity (using the Tetramethylrhodamine ethyl ester, perchlorate probe) using flow cytometry. KMST-6 cell line highlighted selective toxicity towards the cancerous cell lines. CTX-NPs demonstrated significant decrease in cell survival through the inhibition of colony formation (using clonogenic assay) and inhibitory effects on cell migration (using wound healing assay) in U87 cell line. Gene expression analysis using real-time polymerase chain reaction (using the Human Molecular Toxicology Pathway RT² PCR Array) for investigating early cytotoxic effects of CTX-AuPtNPs, showed that genes involved in cellular stress responses were more significant, suggesting that U87 cells activated cytoprotective responses. In addition to the anti-cancer applications, AuPtNPs successfully reduced the 4-nitrophenol to 4-aminophenol at a catalytic rate constant (kcat) of 3.2 x 10-3/sec, demonstrating potential applications in catalysis.