PhoCoil: A photodegradable and injectable single-component recombinant protein hydrogel for minimally invasive delivery and degradation

Abstract

Hydrogel biomaterials offer great promise for three-dimensional cell culture and therapeutic delivery. Despite many successes, challenges persist in that gels formed from natural proteins are only marginally tunable whereas those derived from synthetic polymers lack intrinsic bioinstructivity. Toward the creation of biomaterials with both excellent biocompatibility and customizability, recombinant protein-based hydrogels have emerged as molecularly defined and user-programmable platforms that mimic the proteinaceous nature of the extracellular matrix. Here, we introduce PhoCoil, a dynamically tunable recombinant hydrogel formed from a single protein component with unique multistimuli responsiveness. Physical cross-linking through coiled-coil interactions promotes rapid shear-thinning and self-healing behavior, rendering the gel injectable, whereas an included photodegradable motif affords on-demand network dissolution via visible light. PhoCoil gel photodegradation can be spatiotemporally and lithographically controlled in a dose-dependent manner, through complex tissue, and without harm to encapsulated cells. We anticipate that PhoCoil will further enable applications in tissue engineering and regenerative medicine.

Fig. 1.. PhoCoil structure and stimuli-responsive properties.

PhoCoil hydrogels are physically self-assembled through homopentameric coiled-coil bundle formation. These bundles are held together by noncovalent interactions along the length of the helices, primarily between amino acids at the a and d positions. The noncovalent nature of these associations endows the bulk gel with shear-thinning and self-healing properties in response to applied force. The network degrades in response to 405-nm light due to the cleavage of the peptide backbone within the chromophore of PhoCl. During this process, PhoCl temporarily occupies a red state before the diffusive separation of the two protein halves and subsequent network disassociation. Protein Data Base ID Codes: PhoCl intact (green state), 7DMX; PhoCl cleaved (red state), 7DNA; PhoCl cleaved and dissociated (colorless state), 7DNB; coiled coil, 1MZ9.

Fig. 2.. Viscoelastic properties of PhoCoil gels.

(A) G′ determined from oscillatory rheology time sweeps at 25°C, 5% strain, 10 rad s⁻¹ with varied gel weight percentage. (B) Representative frequency sweep test at 25°C with fixed 5% strain. (C) Representative viscosity profile during shear-sweep test at 25°C. (D and E) Strain-sweep test at 25°C with fixed 10 rad s⁻¹ frequency. (D) Strain crossover value indicated by the arrows in each selected representative strain sweep shown in (E). (F) Cyclic strain sweep test at 25°C. The frequency was fixed at 10 rad s⁻¹, and the strain was held at 500% in gray regions (30 s) and 5% in white regions (600 s). A magnified view of one high strain interval is shown to demonstrate the strain induced gel-sol transition. For all graphs, filled symbols represent G′, and open symbols represent G″. All bars represent the means ± SD of three independently formed gels for each weight percentage. Significance testing was performed using Tukey’s multiple comparisons test. n.s., not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.. Kinetics of PhoCoil gel photodegradation.

(A) Gel softening in response to 405-nm light via oscillatory rheology time sweep. Rheology was conducted at 5% strain and 10 rad s⁻¹ with light intensity set to 10 mW cm⁻² at the gel. G′₀ was defined as the storage modulus immediately before light exposure. Three independent gels were measured for each weight percentage. Data are presented as the means (line) ± SD (shaded area). (B) Control of endpoint stiffness by modulation of light exposure time. Gels (10 wt %) were exposed to constant 10 mW cm⁻², 405-nm light (blue) or periods of 10-min light on, 10-min light off (gray). (C) Control of softening rate by modulation of light intensity. Three independent 10 wt % gels were measured for each intensity of 405-nm light. Data are presented as the means (line) ± SD (shaded area). (D) When normalized for light dosage, experimental data from (C) collapses onto a single curve, indicating that PhoCoil photocleavage follows the same light dose dependency for each intensity. (E) Control of endpoint stiffness by coformulation of PhoCoil with a non–light-responsive network-forming Coil protein. Three independent gels for each formulation were exposed to 10 mW cm⁻², 405-nm light, with the total molar concentration of protein kept constant across formulations. Data are presented as the means (line) ± SD (shaded area).

Fig. 4.. Bulk degradation of PhoCoil gels.

(A) Degradation of varying gel weight percentages in response to increasing time of light exposure. Gels (25 μl) covered with PBS were exposed to 10 mW cm⁻², 405-nm light for 0, 30, 60, or 90 min. Images of the remaining gel in inverted tubes were obtained at the indicated intervals postexposure. (B) Quantification of gel degradation rates. Samples of PBS above each gel were analyzed by BCA assay to determine the protein content as a measure of the percentage of gel degraded at each interval postexposure. Three independent gels were tested for each combination of weight percentage and light exposure time. Data shown are means ± SD.

Fig. 5.. Photopatterning and spatially controlled degradation of PhoCoil gels.

(A) PhoCoil gels can be patterned using photomasks to restrict 405-nm light to desired areas. (B) Photomask-based patterning of grids of varying sizes onto PhoCoil gels. Gels were exposed to 60 min of 10 mW cm⁻², 405-nm light through a grid photomask with light passing through 100-, 50-, or 25-μm-wide squares (top left). Images were obtained in the xy (top) and xz (bottom) planes via confocal microscopy. Scale bars, 100 μm. (C) Intensity in red and green channels was quantified via ImageJ to demonstrate changes in resolution of patterning. Intensity was quantified from the center of a red square to the center of a green line at five random locations. Data shown are means ± SD. (D) 3D reconstruction of the 100-μm grid pattern throughout the thickness of a gel. Scale bar, 250 μm. (E) Spatially controlled degradation of photomask patterned gels. Gels were exposed to 60 min of 10 mW cm⁻², 405-nm light through a photomask and then photographed (true color) or confocal imaged (initial). Gel degradation was allowed to occur in excess PBS, and gels were confocal imaged to visualize degradation (final). Scale bars, 1 mm. (F and G) Light-based degradation of PhoCoil gels through ex vivo tissue. The left half of each gel was covered with a photomask, and the entire gel was placed under 1-mm-thick deli turkey or chicken skin. Gels were exposed to 5 min of high-intensity 405-nm light through the tissue mimic, confocal imaged to visualize the pattern (initial), and left to degrade in excess PBS before final fluorescent imaging. Scale bars, 1 mm.

Fig. 6.. In vivo degradation of PhoCoil gels.

(A) Animals were injected with PhoCoil gel subcutaneously on both the left and ride sides of the dorsal region. IVIS images were taken to detect green (intact gel) and red (cleaved gel) fluorescence before light exposure. Subsequently, the right side of each animal was exposed to 405-nm light to initiate gel degradation. IVIS imaging was repeated after light exposure. (B) Representative IVIS images taken before and after light exposure. Images outlined in green represent green fluorescence, and those in red represent red fluorescence of the injected gels. (C) Quantification of green fluorescence before and after light. (D) Quantification of red fluorescence before and after light. Darker bars represent gels on the left side of each animal, which did not received light. Lighter bars represent gels on the right side of each animal, which did receive light. All bars represent the means ± SD for four animals. Significance testing was performed using Sidak’s multiple comparisons test. n.s., not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7.. Biocompatibility of PhoCoil gels in vivo.

(A) MC38 flank tumors were established, followed by intratumoral injection of PBS, HyStem gel, Coil gel, or PhoCoil gel. Forty-eight hours after injection, the tumor and whole blood were harvested for inflammatory response analysis. (B) Animal weight was monitored immediately after PBS/gel injection and at 24 and 48 hours after injection. All bars represent the means ± SD of n = 4 animals. (C) An inflammatory cytokine array was performed on whole blood collected from each animal to detect potential systemic inflammatory responses. All bars represent the means ± SD of n = 4 animals. Significance testing was done using a one-way ANOVA. n.s., not significant. (D) IHC was performed on tumor tissue to detect potential local inflammatory responses. Scale bars, 1 mm.

Fig. 8.. Viability of PhoCoil-encapsulated fibroblasts.

(A) 10T1/2 fibroblasts are encapsulated in the gel, injected into a well plate, and released from the gel via 405-nm light. (B) Viability of 10T1/2 fibroblasts throughout mock processing (in the suspension, after encapsulation in the gel, and after injection in the gel) for therapeutic cell delivery in PhoCoil gels. All bars represent the means ± SD of three independent gels for each condition. Significance testing was done using Tukey’s multiple comparisons test. (C) Representative confocal microscopy image of Live/Dead analysis of 10T1/2 fibroblasts in the suspension. Scale bar, 250 μm. (D) Representative confocal microscopy image of Live/Dead analysis of 10T1/2 fibroblasts postencapsulation in a 7.5 wt % PhoCoil gel. Image is a maximum projection of a z-stack through a 0.5-mm-thick gel. Scale bar, 250 μm. (E) Representative confocal microscopy image of Live/Dead analysis of 10T1/2 fibroblasts postinjection in a 7.5 wt % PhoCoil gel. Image is a maximum projection of a z-stack through a 0.5-mm-thick gel. Scale bar, 250 μm. (F) Representative confocal microscopy image of Live/Dead-stained 10T1/2 fibroblasts released from a PhoCoil gel. Cells were subjected to encapsulation and injection, followed by release from the gel into a cell culture dish using 405-nm light exposure. Two days following release, cells were stained and imaged to assess their ability to survive and proliferate after such handling. Scale bar, 1 mm. *P < 0.05, **P < 0.01, ***P < 0.001.