(M1030-10-55) Factory-on-a-Pill – a Digital Pill Concept to Deliver Medicines in Space

Purpose: Genetically engineered microorganisms such as Escherichia coli (E. coli) are promising tools for producing biologics like protein therapeutics and vaccines for long-duration space flight. Bioengineered cells can be immobilized within polymeric matrices for stabilization and reduced contamination, which can then be reactivated to produce medicine on site [1]. Our goal is to produce microcarriers to immobilize bioengineered host cells such as E. coli, which can be reawakened to produce target proteins that can be suspended in 3D-printed pills.

Methods: Lysogeny broth (Fisher Bioreagents), Pluronic® F127 (D-BASF), and alginic acid sodium salt (Acros Organics) were used to formulate the hydrogel for the 3D-printed tablets. Multiple formulations were crafted, with formulation 7 being the most stable with 25% Pluronic-F127 and 5% alginic acid sodium weight by volume. The formulation was stirred in the refrigerator for 24 hours then added to 3 mL pneumatic syringes for 3D printing using the BioX 3D printer. The pressure ranged from 200 to 400 kPa, the infill density ranged from 25% to 40%, and the infill shape was concentric. Tablets were incubated at 37°C, 100 rpm for 24 hours to assess for stability and any physical changes in consistency, color, and shape. Both placebo and E. coli pills were printed, after which a viability test was conducted for seven days. Absorbance was measured at 24, 48, and 72 hours at 600 nm. 72 hour data is shown in Chart 1.

Results: Formulations 1 to 6 were not printable due to its liquid consistency. Formulation 7 was more viscous than formulations 1 to 6, hence its printability. F7 was used to print different sized pills (9 mm to 15 mm), thickness (1 mm to 3 mm), infills (25% - 40%), and infill patterns (Grid, Honeycomb, Rectilinear, Linear, Concentric, Gyroid) (Table 1). 12x12x2 mm pills with a concentric infill pattern showcased the best stability. E. coli growth was consistent throughout the viability study, showcasing the survivability of E. coli in our formulation. The absorbance range for the E. coli was 0.30 to 0.47, which steadily increased every absorbance. The absorbance range for the placebo pills was 0.09 to 0.38, with no consistency between the increase and decrease of the absorbance (Table 2).

Conclusion: Formulation 7 was successfully printed and remained consistent during incubation, though further alterations may need to be done to ensure the proper solidification of Formulation 7. Regardless, the printed tablets present a stable environment for E. coli growth, though contamination did occur with the placebo pills. In order to alleviate the contamination, we will use ultraviolet light and print under a biosafety cabinet to ensure sterilization. Moreover, further studies to assess protein production will be conducted to ensure that immobilized E. coli within the pill can express protein in order to produce biologics. Since biologics, like vaccinations and insulin, need specific storage conditions, our proposed pills can help promote future aerospace research by providing astronauts with the necessary medicine when they require it.

References: [1] Guo, Y.; Wang, G.; Chen, H.; Zhang, S.; Li, Y.; Guo, M.; Liu, J.; Chen, G. Continuous Fermentation by Lactobacillus bulgaricus T15 Cells Immobilized in Cross-Linked F127 Hydrogels to Produce ᴅ-Lactic Acid. Fermentation 2022, 8,360.

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

Table 1. 3D-Printing Parameters Applied on F7

Table 2. Pill Viability Data Overview

Chart 1. Pill Viability 72 Hour Data