Bioprinting of a Probiotic Time-Dependent Oral Colon Delivery System for Bacteriotherapy

Gut dysbiosis results from unique interactions between environment and host factors and is increasingly being related to a wide range of pathological conditions, not limited to the gastrointestinal tract1,2. Restoring microbial balance through colon targeted delivery of probiotics selected based on each patient’s needs has accordingly been described as a strategy for effective management of several diseases3. However, techniques traditionally used for manufacturing oral solid dosage forms, such as tableting and coating, not only may expose bacteria to mechanical stress and elevated temperatures but also would be unfit for customized probiotic administration4. To overcome these limitations, 3D bioprinting is here explored as an innovative technique for fabrication of a time-dependent oral colon delivery system containing model probiotic Lacticaseibacillus paracasei. Disintegrating cores were fabricated from pastes containing paracetamol as an analytical tracer, and soluble Mannogem®XL as the filler. The bacterial pellet was incorporated just prior to printing. An inner shell, aimed at delaying the onset of release until colon arrival, was printed using hydroxypropyl cellulose (HPC, Klucel®LF), while an outer one consisted of an enteric polymer, hypromellose acetate succinate having a dissolution pH threshold of 6 (HPMCAS, AQOAT®AS-MF), was added to overcome variable gastric emptying time. Bioprinting was performed by Discovery™ Gen.5 system (RegenHU, CH), combining extrusion and thermoplastic printheads. Following trial-and-error setup of the printing substrates and parameters, formulations exhibiting good rheological properties were compounded, and the process was run without disruption. Notably, the probiotic-containing core and inner HPC shell were fabricated in a single printing step, followed by oven-drying (<35°C) and addition of the enteric outer shell in a subsequent step. The consistent extrusion performance was pointed out by consistency of the overall characterization data. Release tests demonstrated that gastroresistance was achieved and, as desired, a delay phase was brought about by polymer swelling/erosion. L. paracasei viability was not harmed by compounding, 3D printing and drying, thus ultimately highlighting feasibility of multilayered dosage forms for possible precision bacteriotherapy using 3D bioprinting technique. References and Citations 1. Nie, P.; Li, Z.; Wang, Y.; Zhang, Y., Zhao, M.; Luo, J.; et al. Gut microbiome interventions in human health and diseases. Med. Res. Rev. 39, 2286–2313 (2019) 2. Schluter, J.; Peled, J.U.; Taylor, B.P.; Markey, K.A.; Smith, M.; Taur, Y., et al. The gut microbiota is associated with immune cell dynamics in humans. Nature, 588, 303–307 (2020) 3. Nagayama, L.; Gogokhia, L.; Longman, RS. Precision microbiota therapy for IBD: premise and promise, Gut Microbes, 17, 1, 2489067 (2025). 4. Bosch, B.; Moutaharrik, S.; Gazzaniga, A.; Hiippala, K.; Santos, H.A.; Maroni, A.; Satokari, R. Development of a time-dependent oral colon delivery system of anaerobic Odoribacter splanchnicus for bacteriotherapy. Eur. J. Pharm. Biopharm, 190, 73-80 (2023).