Novel pH triggered fluorescent mesoporous silica nanoparticles for theranostic applications in cancer intervention

Abstract

Cancer is set to become the leading cause of death globally and is the biggest impediment to increased life expectancy in the 21st century, as the global incidences and mortality rates are forever increasing. The ability to simultaneously target, detect and treat cancer would reduce patient mortality as a result of early disease detection and immediate targeted treatment. Two highly studied phytochemicals namely curcumin and lawsone were selected for use in this study owing to their established anti-cancer properties and their innate fluorescence; however, their clinical applications are hindered by several limiting factors. This study aimed to improve the biopharmaceutical properties of curcumin and lawsone to further exploit their purported therapeutic properties by synthesising a novel therapeutically stable molecule with appropriate bioavailability, chemotherapeutic properties and innate fluorescence. In addition, the new combined molecule was designed to have pH-responsivity based on the known acidification of the extra cellular matrix during malignant transformation, enabling pH specific cancer targeting. A novel molecule (2,2'-((((1E,3Z,6E)-3-hydroxy-5-oxohepta-1,3,6-triene-1,7-diyl)bis(2- methoxy-4,1-phenylene))bis(oxy))bis(naphthalene-1,4-dione) was synthesised through a Williamson ether synthesis reaction between curcumin and lawsone and was termed CurNQ. The structure of CurNQ was evaluated through 1H and 13C NMR, HRMS-ESI and FTIR and physiochemical characterisation was performed through PXRD and DSC. The loading of CurNQ onto a nanoplatform aimed to allow for enhanced delivery and enable in vitro and in vivo imaging and detection applications. To this end, mesoporous silica nanoparticles (MSN) were synthesised (size = 108 d.nm, Zeta potential -42 mV, PDI= 0.150) and were impregnated with CurNQ, forming the novel nanosystem MSN_CurNQ. The MSN nanoplatform aimed to enable passive tumour targeting owing to the EPR effect and for sustained release of CurNQ at the tumour site. CurNQ was shown to have pH specific solubility, allowing for pH mediated cancer targeting owing to the Warburg effect. The saturation solubility of CurNQ increased xii from 11.15 µM at pH 7.4 to 20.7 µM at pH 6.8. CurNQ release from the MSN mimicked this solubility switch whereby after 96 hours 31.5% of CurNQ was released at pH 7.4 compared to 57% release at pH 6.8. The large statistically significant pH specific shift in solubility (p=0.000) coincides with the shift in pH that occurs upon malignant transformation and allows for potential cancer targeting applications. The fluorescent nature of MSN_CurNQ was evaluated using confocal and fluorescent microscopy. The microscopy images demonstrate MSN_CurNQ to have highly precise, distinctive high intensity innate fluorescence, with a high target to background ratio, low fluorescent blur, a long fluorescent lifetime and fluorescent stability, all of which demonstrate MSN_CurNQ to be a potentially powerful diagnostic and imaging agent. CurNQ and MSN_CurNQ induced cytotoxicity to several cancer cell lines. CurNQ reduced cell viability to the ovarian cancer cell lines OVCAR-5 and SKOV3 to below 50% with a potent IC50 value of 4.048 µM observed for the OVCAR-5 cell line. Additionally, MSN_CurNQ induced a reduction in cell viability to below 50% in OVCAR 5, CACO-2, CHLA and MCF-7 cell lines demonstrating chemotherapeutic potential to a variety of cancer cell types. Furthermore, both CurNQ and MSN_CurNQ were less toxic to the healthy fibroblast cell line 3T3 compared to the toxicity displayed towards several cancer cell lines (p=0.001, 0.000); thus, demonstrating cancer selective toxicity further extending its novelty and its appropriateness as a potential chemotherapeutic agent. The promising in vitro results prompted the commencement of a preclinical trial in a female MF-1 nude mice model. The preclinical trial demonstrated CurNQ and MSN_CurNQ to be well tolerated, inducing low overall toxicity and to be safe at an administered dosage of 10 mg/kg. OVCAR-5 tumour xenografts were successfully established in MF-1 nude mice and the potential efficacy of CurNQ and MSN_CurNQ as a chemotherapeutic agent was evaluated. Preliminary results suggested CurNQ and MSN_CurNQ may induce tumour necrosis and may reduce tumour growth rates; hence these results were highly promising and warrant the continuation of further in vivo studies. Indeed, nano-enabled CurNQ acts as a promising theranostic nanosystem potentially allowing for simultaneous cancer targeting, detection and treatment.