(T1030-07-44) Development of a 3D-Printed Hydrogel Pill Encapsulating Bioengineered Microorganisms for On-Demand Therapeutic Protein Production

Purpose: This study aims to develop a 3D-printed hydrogel pill capable of delivering therapeutic proteins by immobilizing bioengineered Escherichia coli. These hydrogel-based tablets, designed for long-term room-temperature storage, offer a promising platform for the on-demand production and delivery of biologics, particularly large molecules for cancer treatments.

Methods: The hydrogel was formulated using Lysogeny Broth (Fisher Bioreagents), Pluronic® F127 (D-BASF), and alginic acid sodium salt (Acros Organics). Seven formulations were evaluated, and Formulation 7 (F7) was selected based on optimal printability and rheological performance. F7 was printed using the BioX® 3D printer, and its rheological properties were analyzed using a Modular Compact Rheometer. FTIR spectra of excipients and placebo tablet was conducted using a Cary 630 FTIR spectrometer with a Cary FTIR ATR fitted with a single-bounce diamond-coated ZnSe internal reflection element. Bioengineered E. coli K12 MG1655 expressing TNF-alpha was incorporated into the hydrogel. Tablet swelling was assessed in water over 45 minutes using the formula: Sr = (Ww − Wd) / Wd × 100, and water loss was measured over 72 hours at 37 °C. E. coli pellets were obtained via incubation and centrifugation, then mixed with F7 hydrogel for tablet bioprinting. Both placebo and bacteria-loaded tablets were stored at room temperature and 37°C for 28 days. Viability and protein expression were monitored throughout the study period. Bacterial encapsulation was evaluated using Scanning Electron Microscopy (JEOL JSM-5600). TNF-alpha expression was quantified using a BioLegend® ELISA Max™Deluxe kit, and cytotoxicity was assessed using B16 mouse melanoma cells with control E.coli and E. coli K12 . B16 cells were cultured in a transwell plate (4 x 10⁵ cells/well) for 24 hours. Cytotoxicity was observed under microscopy and confirmed after the CCK-8 assay. JMP® was used for statistical analysis.

Results: The hydrogel formulation (F7) exhibited temperature-sensitive viscosity ranging from 18,000 mPa·s at 10 °C to 360,000 mPa·s at 30 °C, with storage modulus (G’) exceeding loss modulus (G’’), confirming gel-like behavior suitable for 3D printing. F7 printed tablets showed printability and shape fidelity (Figure 1). The swelling ratio reached 1.2 within 45 minutes, and water loss was estimated at 73% after 72 hours at 37 °C. FTIR showed no interaction between components. Viability assays demonstrated consistent E. coli growth in hydrogel tablets over 28 days at room temperature (OD600: 0.45–0.6), while placebo tablets remained below 0.1. However, E.coli growth stopped after 10 days for the tablet stored at 37°C. TNF-alpha expression from the hydrogel tablets ranged from 0.45 to 117 pg/mL over the study period for room temperature stored tablets. For the tablets stored at 37°C, protein expression ranged between 48 and 55 pg/mL for the first 10 days of the study and then no protein was released for the rest of the study (Figure 2). Transwell plate cytotoxicity assays indicated significant inhibition of B16 melanoma cells after 24 hours, compared to untreated cells and cells exposed to non-engineered E. coli (Prob > F = 0.0222*). Microscopy images confirmed reduced melanoma cell density following protein treatment (Figure 3).

Conclusion: In conclusion, the F7 hydrogel formulation supports successful 3D bioprinting of microorganisms, stable bacterial encapsulation, and sustained production of therapeutic proteins. The printed tablets maintain function for 28-day room-temperature storage. This 3D-printed platform offers a promising solution for on-demand biomanufacturing of protein-based therapeutics.

Acknowledgements: The authors declare no financial conflict of interests. This research was partially sponsored by NIH/FDA (1R01FD007456-01) and NASA, #80NSSC20M0101 (350221329A).


Hydrogel tablet printability in terms of size, thickness, infill pattern, and infill density


E.coli viability and TNF-alpha expression from tablets: a) Bacteria viability in tablets stored at room temperature b) Protein expression from tablets stored at room temperature c) Bacteria viability in tablets stored at 37°C c) Protein expression from tablets stored at 37°C


Cytotoxicity study of secreted TNF-alpha against B16 mouse melanoma cells: a) Non-treated B16 cells b) B16 cells treated with non-engineered E.coli c) B16 cells treated with bioengineered E.coli