Light-guiding hydrogels for cell-based sensing and optogenetic synthesis in vivo

Abstract

Polymer hydrogels are widely used as cell scaffolds for biomedical applications. While the biochemical and biophysical properties of hydrogels have been extensively investigated, little attention has been paid to their potential photonic functionalities. Here, we report cell-integrated polyethylene glycol-based hydrogels for in-vivo optical sensing and therapy applications. Hydrogel patches containing cells were implanted in awake, freely moving mice for several days and shown to offer long-term transparency, biocompatibility, cell-viability, and light-guiding properties (loss: <1 dB/cm). Using optogenetic, glucagon-like peptide-1 (GLP-1) secreting cells, we conducted light-controlled therapy using the hydrogel in a mouse model with type-2 diabetes and attained improved glucose homeostasis. Furthermore, real-time optical readout of encapsulated heat-shock-protein-coupled fluorescent reporter cells made it possible to measure the nanotoxicity of cadmium-based bare and shelled quantum dots (CdTe; CdSe/ZnS) in vivo.

Keywords: Biosensor; Hydrogel; Optical waveguide; Optogenetics; Photomedicine; Synthetic biology.

Figure 1

Schematic of a light-guiding hydrogel encapsulating cells for in vivo sensing and therapy. The cells in the implanted hydrogel produce luminescence in response to environmental stimuli (sensing) and secrete cytokines and hormones upon photo-activation (therapy). The light guiding hydrogel establishes a bi-directional optical communications with the cells, allowing real-time interrogation and control of the biological system in vivo.

Figure 2

Characteristics of hydrogels. (a) Photographs of PEG-based hydrogels prepared by using 10% w/v PEGDA solution with different MW’s of 0.5, 2, 5, and 10 kDa, respectively. Scale bar, 1 cm. (b) The optical attenuation spectra of PEG hydrogels prepared with different MW’s of PEGDA. (c) Rectangular-shaped 0.5- and 5-kDa hydrogels with a thickness of 1 mm. Scale bar, 5 mm. (d) Swelling ratios of PEG hydrogels. The swelling ratio was calculated by dividing weight of swollen hydrogel by weight of dried hydrogel (n = 3). (e) Mechanical flexibility of the PEG hydrogel (5 kDa, 10%). (f) Demonstration of total internal reflection within the slab hydrogel.

Figure 3

Light-guiding properties of fiber-optic hydrogels. (a) A setup for coupling light into a hydrogel waveguide via a multimode fiber. (b) Photographs showing light coupling to a hydrogel Top, a hydrogel before light coupling; middle, a hydrogel after light coupling; and bottom, a pseudo color image for spatial profile of scattered light. (c) Schematic of the setup for measuring the collection efficiency. A fluorescent sample (green) was placed in contact with hydrogels with varying lengths (left) or at the equivalent distances from a multimode fiber (right). (d) Magnitude of fluorescence collected into the optical fibers with and without the hydrogels. Dashed lines, curve fit with 1/L² and 1/L dependence for the hydrogel and optical fiber alone, respectively. (e) The ratio of fluorescence with and without the hydrogels. The dashed line represents a linear regression (R² = 0.98). (f) The optical attenuation spectra of the hydrogels at various cellular density levels. Inset shows a phase-contrast micrograph of the hydrogel. Scale bar, 50 µm. (g) Average optical attenuation of a hydrogel with 1 × 10⁶ cells/cm³ in a spectral range of 450–500 nm. The dashed line shows an exponential fit (R² = 0.96).

Figure 4

Hydrogel implants in vivo. (a) Schematic of a fiber-pigtailed hydrogel waveguide implanted in a mouse. (b) A hydrogel-implanted mouse in a freely moving state. Blue light (491 nm) was coupled. (c) Photographs showing the light scattering profiles from an optical hydrogel implant (left) and from an optical fiber (right). (d) Axial profiles of the magnitude of scattered light from the hydrogel implant (blue) and the optical fiber only (black). (e) Fluorescence images of hydrogel implants immediately after taken out of mice at 3 days and 8 days after implantation, in comparison to control (Day 0; prior to implantation). Live cells emit green fluorescence from a membrane-permeable live-cell staining dye, calcein-AM, and dead cells are identified by red fluorescence from ethidium bromide in the cell nuclei. Scale bar, 50 µm. (f) Long-term viability of the encapsulated cells in vivo. Error bars are standard deviations (n = 6 each). (g) Change in optical transmittance of the hydrogel implants in vivo. (h) H&E histology images of the skin tissues examined at 8 days after implantation, showing (i) dermis, (ii) panniculus carnosus, (iii) subcutaneous loose connective tissue layer, (iv) newly formed connective tissue layer. In the magnified image (right), arrows indicate red blood cells in blood vessels. Scale bar, 100 µm.

Figure 5

Cell-based sensing of nano-cytotoxicity of quantum dots. (a,b) Fluorescence images of the sensor cells in hydrogels in vitro at two days after adding CdTe and CdSe/ZnS Q-dots, respectively, into the medium. Scale bar, 20 µm. (b) The magnitude of green fluorescence from the hydrogels, measured through the pigtail fibers. (c) In vivo measurement of the fluorescence signals from the sensing cells in hydrogels implanted in live mice. Q-dots were administered by intravenous injection 24 hours after the hydrogels were implanted. (d) Fluorescence images of the hydrogels extracted from the mouse at day 3. Scale bar, 20 µm. (e) Comparison of the GFP fluorescence measured fiber-optically in vivo (left) and fluorescence microscopy ex vivo (right).

Figure 6

Optogenetic therapy in a mouse model of diabetes. (a) Fluorescence calcium-level imaging of optogenetic cells in a hydrogel waveguide in vitro. Upon delivering blue light (455 nm) through the fiber for 10 s at 1 mW, the fluorescence from an intracellular calcium indicator, OGB1-AM increased significantly. Scale bar, 20 µm. (b) Time traces of intracellular calcium signals from various cells marked in (a). (c) Concentrations of active GLP-1 in the medium of hydrogels with (ON) and without (OFF) providing the activation light. (d) Level of GLP-1 in blood plasma measured in vivo at 2 days after light exposure. (e) Blood glucose levels in the chemically induced diabetic mice with and without delivering activation light. Error bars, standard deviations (n = 4).