(W0930-04-26) A Novel Poly(N-isopropylacrylamide)-based Thermoresponsive Bioink for DLP Bioprinting

Purpose: Thermoresponsive polymers have been traditionally used in drug delivery, tissue engineering, and other biomedical applications. They possess the ability to undergo reversible phase transition in response to temperature changes. This unique property of transitioning from sol to gel leading to changes in their physical state makes them attractive for bioprinting applications. However, current challenges in formulating thermoresponsive polymers include poor mechanical properties, limited printability, and poor cell viability. Poly (N-isopropylacrylamide) (PNIPAm) is one such thermoresponsive, biocompatible polymer widely used in drug delivery and cell sheet engineering, undergoing its reversible phase change at a key lower critical solution temperature (LCST). DLP (Digital Light Processing) printing is a type of 3D printing that uses a digital projector to selectively cure a liquid photopolymer resin or bioink layer by layer to create a 3D object. The projector illuminates the resin with a specific pattern of light, which causes polymerization to occur and solidify in the desired shape. DLP printing offers high printing speeds, high resolution, and the ability to produce complex geometries with intricate details, making it an attractive method for 3D printing. We report here the development of a novel thermoresponsive and biocompatible photopolymerizable liquid bioink prepared using PNIPAm, for DLP printing to yield hydrogels that can be used for various biomedical applications including 3D cell culture and drug delivery. Our goal was to print high resolution structures in the most efficient manner and characterize the resultant hydrogels for their physical and in vitro properties. Building on work previously published by our lab, we also plan to utilize these hydrogels for high-throughput generation of cell spheroids.

Methods: The bioink was prepared by dispersing N-Isopropylacrylamide (NIPAm), N,N -Methylenebis(acrylamide) (Bis), Tartrazine, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) in de-ionized water. Two full factorial experiments were conducted to determine the optimal formulation with clear, reversible phase transition at an LCST close to 37°C and minimal bleaching. The LCST was determined by derivatization of UV-vis absorbance measurements of the formulation at different temperatures. The thermoresponsive properties of the printed hydrogel were assessed using shrinking and swelling studies and contact angle measurements. Mechanical properties above and below the LCST were also studied using rheology with a parallel plate configuration and compression testing (TA Instruments HR30). LCST-dependent release kinetics of bovine serum albumin (model macromolecule drug) from the hydrogel was also evaluated in PBS (pH 7.4), while small molecule release kinetics are currently under similar evaluation. Further, in vitro validation was done by culturing single- and multi-layer spheroids using a combination of HeLa ovarian sarcoma and human dermal fibroblast (HDF) cells.

Results: A photopolymerizable and thermoresponsive liquid bioink using PNIPAm was successfully synthesized. The optimized 3D printed formulation demonstrated sharp, reversible and temperature-dependent phase transition at an LCST of 36.8°C (Figure 1). Several different structures including microwells, kidney, heart, discs, spheres and cylinders were printed successfully using the novel bioink (Figure 2). Reswelling studies confirmed that the hydrogels swelled to 100% of its initial weight within 4 hours. Contact angle studies confirmed a decrease in contact angle from 44.4° at 25°C to 71.0° at 37°C (Figure 3). Mechanical testing above the LCST demonstrated that the hydrogel become brittle and solid like with little to no elastic deformation, while below the LCST they were elastic and semi-solid like. Leaching cytotoxicity showed no marked cell death up to 7 days. Cell spheroids using HeLa and HDF cells were successfully cultured within 3D printed microwell arrays up to 7 days. BSA release studies demonstrated the release of 100% of the encapsulated drug within 7 days at 25°C, and 65% in 21 days at 37°C, an interesting reversal of the expected release pattern caused by a specific BSA-PNIPAm interaction.

Conclusion: We have successfully formulated a novel, reversibly thermoresponsive and cytocompatible polymer photoink for use with DLP bioprinters, capable of printing an array of different structures with high print fidelity. The formulation demonstrates excellent thermoresponsive capabilities and supported long-term 3D cell culture indicating their utility for a diverse range of medical applications.

References: D. Dhamecha, D. Le, T. Chakravarty, K. Perera, A. Dutta, J.U. Menon, Fabrication of PNIPAm-based thermoresponsive hydrogel microwell arrays for tumor spheroid formation, Mater. Sci. Eng. C. 125 (2021) 112100. https://doi.org/10.1016/j.msec.2021.112100.

Microwell arrays (12x12 arrays of wells of 800 um diameter) imaged below (i) and above (ii) the formulation's LCST of 36.8°C. Note the change in size as a result of the hydrogel's thermoresponsive swelling/shrinking. These arrays may be used for high-throughput culture of cell spheroids for a variety of screening applications.

Images of miniature human kidney (i) and heart (ii; without pulmonary vasculature) printed with our optimized formulation, demonstrating its versatility for printing custom-shaped drug delivery implants or other devices.

Contact angle studies showing the thermoresponsive hydrophilicity/hydrophobicity of our formulation: images below (i) and above (ii) the formulation's LCST of 36.8°C, and graphical representation of the same (iii; n=3).