Developing Upconverting Nanoparticle-Based Force Sensors for In Vivo Gastrointestinal Imaging

Mechanical forces play a fundamental role in a myriad of biological processes such as stem cell differentiation, cardiovascular health, cancer progression, and digestion. The complex interplay of mechanical forces is important for understanding disease development, but current biological force measurement techniques are limited: <i>ex vivo</i> techniques such as atomic force microscopy and traction force microscopy do not measure forces in accurate physiological conditions, while <i>in vivo</i> measurement techniques like stents and catheters cause uncomfortable colon distension and risk damage or rupture. Nanoscale markers such as resonant energy transfer-based molecular tethers, oil droplets, and quantum dots also lack the robust signal to overcome a rapidly changing biological environment.<br/>Here, we demonstrate upconverting nanoparticles (UCNPs) as promising minimally invasive optical force sensors, conjugated to polydimethylsiloxane (PDMS) pellets for <i>in vivo</i> gastro-intestinal tract imaging. UCNPs are photostable, absorb in the near-infrared tissue transparency region, can be functionalized to target specific biological structures, and do not require toxic heavy metals like cadmium for synthesis. We synthesize monodisperse 20-nm diameter cubic phase NaYF4:Yb, Er@NaYF4 UCNPs, then use dual confocal-AFM microscopy to exert forces on single particles and determine their dynamic range of mechanosensitivity in the nanoNewton to microNewton force regime. Next, we cast PDMS mimics of mouse fecal pellets to create a colonic probe. We then embed the UCNPs in silica microspheres functionalized with thiol groups to enhance their signal, functionalize the PDMS with alkene groups via allyltriethoxysilane, then conjugate these UCNP-SiO2 microspheres to the PDMS via thiol-ene click chemistry. We calibrate the spectral response of these UCNP-PDMS probes using a custom-built air pressure microscope chamber that suspends the UCNP-PDMS pellet on a wire to radially apply pressure for colon force detection, demonstrating that red-to-green emission ratio changes repeatably over multiple compression and decompression cycles from 0 to 15 Pa applied pressure. Finally, we deploy these pellet probes in excised mouse colons from both wild type and Etv-/- knockout mice to image and quantify forces in healthy colon pumping and irregular colon pumping, respectively. We compare the time-averaged force and location-dependent force exerted by the colons, as well as total time for pellet transition to demonstrate that our UCNP mechanosensor is able to distinguish between biological phenotypes with bright, robust optical signal that can penetrate colon wall tissue without damaging it. This platform provides a straightforward method for imaging intraluminal force dynamics in a range of hollow organs, such as the stomach or heart cavities.