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Colorectal cancer (CRC) is a cancer type with a high incidence among the adult population. Standard treatments risk harming surrounding healthy tissues due to non-specificity and causing systematic side effects, highlighting the need for new therapies.  A tumor-specific and less invasive approach could involve a two-step administration via the oral route. The first step is the administration of cancer-specific antibodies. The second step involves the administration of superparamagnetic iron oxide nanoparticles (SPIONs), which serve as both magnetic resonance imaging contrast agents and thermal therapeutic agents. These SPIONs bind to the antibodies via strain-promoted Azide-Alkyne Click Chemistry reaction (SPAAC), enabling theragnostic applications. However, agglomeration and size of SPIONs in biological media must be addressed for effective targeting at the tumor site in the intestinal epithelial wall. We therefore aim to reduce the particle agglomerate size in cell culture media (CCM) while maintaining the SPAAC click-ability, and additionally to perform an in vitro cell study for this purpose.

In this project, silica coated SPIONs were functionalized according to a one- or two-step method to obtain the click-handle dibenzocyclooctyne (DBCO) on the nanoparticle surface. In addition, either carboxylic acid (COOH) or polyethylene glycol (PEG) containing molecules were added for stabilizing particle agglomeration. Eight different functionalized particles were evaluated for SPAAC reaction using fluorescence. One particle was selected for a pilot pre-targeting study in Caco-2 cells using azide-modified antibodies. Inductively coupled plasma optical emission spectroscopy was carried out for evaluation of SPAAC reaction in cell models.

Both COOH and PEG showed reduction of hydrodynamic diameter of DBCO functionalized SPIONs in water and in CCM. The smallest agglomerate size in CCM was obtained by the DBCO-PEG/mPEG-NP 1:1.5:1 (451 nm), but it failed to undergo SPAAC reaction. Out of the particles able to undergo SPAAC reaction, the COOH/DBCO-NP 50:2 had the smallest hydrodynamic diameter (968 ± 192 nm). Therefore, this particle was chosen for cell study. Surprisingly, the results showed that metal concentrations were higher in the control group treated with SPIONs compared to those treated with COOH/DBCO-NP 50:2.

PEG-chain linkers showed better hydrodynamic diameter reduction effect in CCM than COOH. One explanation can be due to less protein adhesion. Particles functionalized with PEG linkers showed weaker click chemistry properties, which makes them less suitable for in vivo pre-targeting. This could be due to steric hindrance by PEG or less yield of functionalization. From these findings we can conclude that nanoparticle hydrodynamic diameter reduction is possible with both COOH and PEG. However, click chemistry properties were lost during PEG-functionalization. Also, the cell model for evaluating the SPAAC reaction and pre-targeting approach needs to be further developed based on the outcome of this project.