Targeted Biodegradable Near-Infrared Fluorescent Silica Nanoparticles (FSNs) for Colorectal Cancer Imaging

Introduction: Colorectal cancer (CRC) is the third leading cause of cancer-related death in the United States. Somewhat paradoxically, it is also easily treatable when caught early, with a 5-year survival rate of about 90%. However, many cases are not caught until the disease has invaded the mucosa; for these cases, the 5-year survival rate is only 14%. Despite robust screening recommendations, many CRC cases are caught too late for lifesaving intervention. For this reason, an imaging agent capable of detecting otherwise undetectable lesions at a molecular level would be a life-changing technology. Fluorescent endoscopy combines the widefield real-time imaging characteristics of traditional endoscopy with the sensitivity and specificity afforded by fluorescent nanomaterials. Specifically, fluorescent silica nanoparticles (FSN) are an ideal choice for fluorescent endoscopy due to their straightforward synthesis, high quantum yield, biocompatibility, and facile surface modification with targeting molecules. In this study, we synthesized near-infrared FSNs conjugated with a selective CRC-targeting antibody and evaluated their use as a CRC imaging agent in vitro and in vivo.

Materials and

Methods: NIR-FSNs were synthesized via a modified Stöber reaction following covalent linking with NIR fluorescent CF800 dye. Briefly, CF800-(3-Mercaptopropyl) trimethoxysilane was combined with tetraethyl orthosilicate (TEOS) in a mixture of isopropanol and ammonium hydroxide. Particle size (50, 100, and 200 nm) was controlled by changing the reaction time. The particles were then washed in ethanol and PEGylated. Finally, the PEGylated, CF800-FSNs were conjugated to selective biomarkers of CEA antibodies through an EDC-NHS coupling reaction. The size and shape of particles were determined through transmission electron microscopy. Their hydrodynamic size and polydispersity index were measured by dynamic light scattering. Particles’ NIR fluorescence was measured using a PEARL imaging system. Biodegradability was studied by incubating the particles in simulated body fluid and imaging them by TEM. Cell uptake of CEA-FSN was performed in HT29 cells and HCT 116 cells to validate preferential uptake by CEA-expressing cells. An additional uptake experiment was performed in HT29 cells using targeted CEA-FSN and untargeted FSN. Targeting in vivo studies were conducted by intravenous injection of PEG-FSN and CEA-FSN (10 mg/kg) in CRC xenograft mice and imaged by PEARL at various time points (0, 1, 4, 8, 24, 48 h). Finally, target-specific accumulation of CEA-FSN after topical application to polyposis in the rat colon (PIRC) rat models was confirmed via ex vivo imaging. Statistical analysis was performed using a student’s t-test and considered significant at p< 0.05.

Results, Conclusions, and Discussions: Uniform and monodisperse NIR-FSNs were successfully synthesized. 50 nm particles were completely degraded in a biological media after 7 days, while larger particles (100 and 200 nm) were not. The 50 nm FSNs exhibited no cytotoxicity at high concentrations. The particles were shown to be successfully conjugated to PEG or CEA-antibodies through a shift in hydrodynamic diameter. They exhibited strong NIR fluorescence signals (at least 2.2 times higher than the dye-only control). Western blotting confirmed that HT29 cells express CEA, while HCT116 cells do not. Unsurprisingly, when both cell lines were treated with CEA-FSN, HT29 cells showed significantly higher particle uptake than HCT116 cells. Further target specificity of our particles was confirmed by the enhanced uptake of CEA-FSN compared to untargeted FSN in HT29 cells. These results indicate the CEA-FSNs can selectively target CEA expression and effectively detect CRC cells. Through in vivo imaging, we found CEA-FSN selectively accumulated in CEA-positive HT29 tumors at 1 and 4 hrs post-injection, with at least a two-fold increase in fluorescence intensity compared to CEA-negative HCT116 tumors. After the presence of polyps in PIRC rats was validated through endoscopy, CEA-FSNs were applied topically to the intestine. Colocalization of polyps (white-light endoscopy) and nanoparticles (NIRF signals) was confirmed from the excised intestine tissues. Immunofluorescence imaging validated the colocalization of CEA, FSN, and a CRC maker (β-catenin). The results indicate that the CEA-FSN can be used as a surrogate imaging probe for fluorescent endoscopy, allowing for earlier detection of CRC and, ultimately, more successful treatment.

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