Theranostic CEA-Affimer functionalised silica nanoparticles allow specific in vitro fluorescent imaging of colorectal cancer cells.


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Yazan S. Khaled1,Shazana Shamsuddin1,Jim Tiernan1,Mike McPherson1,Thomas Hughes1,Paul Millner1,David G. Jayne1
1University of Leeds

Abstract

Background

Fluorescent laparoscopic imaging of primary colorectal tumours and lymph node metastases would potentially facilitate intra-operative stratification of surgical resections and improve patient outcomes. Nanoparticulate platforms offer many advantages in terms of intra-operative imaging and biosensing for diagnostic purposes. Nanoparticles can be loaded with diverse reagents and then addressed to the correct tissue provided a specific biomarker molecule exists to which an antibody or Affimer (artificial binding protein) can be developed. We aimed to develop a fluorescent silica nanoparticles targeted against colorectal cancer (CRC) using an anti-carcinoembryonic antigen (CEA) Affimer (Aff). 

Method

Anti-CEA and control Myoglobin-Affimer binders were subcloned into the expressing vector pET11 followed by transformation into BL21-Starâ„¢-(DE3) E.coli. The photosensitiser Foslip (soluble analogue of 5,10,15,20-Tetra(m-hydroxyphenyl)-chlorin) was incorporated into the core of silica nanoparticles using water-in-oil microemulsion technique. Anti-CEA or control-Affs were conjugated to silica nanoparticles surface using sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC) chemical linker. Binding of CEA-Aff or control nanoparticles to CRC cells (LoVo, LS174T and HC116) was quantified in vitro using confocal microscopy. 

Results

The molecular weights of the obtained band of Affimers were ~12.5KDa whilst the diameter of functionalised silica nanoparticles was ~80nm. CEA-Affimer targeted nanoparticles demonstrated 9.4, 5.8 and 2.5 fold greater fluorescence than control in, LoVo, LS174T and HCT116 cells respectively (p<0.002) for the single slice analysis. A similar pattern of successful CEA-targeted fluorescence was observed in the maximum image projection analysis (p<0.0002). There was no significant difference in fluorescence for CEA-Affimer vs. CEA-antibody targeted nanoparticles. 

Conclusion

We are the first to demonstrate that Foslip-doped silica nanoparticles conjugated to anti-CEA Affimers via SMCC allows tumour cell-specific fluorescent targeting in vitro, and have shown sufficient promise to justify testing in an animal model of colorectal cancer. CEA-Affimer appears to be a suitable targeting molecule to replace CEA-Antibody. Targeted silica nanoparticles loaded with Foslip photosensitiser are now being optimised to drive photodynamic killing.