3D Bioprinting of an Oral Colon Delivery System for Precision Bacteriotherapy

Abstract

Objectives: A customizable 3D-bioprinted core-in-shell platform was developed for time-dependent oral colon delivery of live microorganisms. The system conveyed Lacticaseibacillus paracasei as a model bacterial species within a monolithic core, which was surrounded by a swellable hydroxypropyl cellulose barrier, imparting a lag phase of programmable duration, and by an enteric outer layer, protecting the dosage form during unpredictable gastric residence.

Methods: Pastes of different compositions were investigated to shape the core. Core and core-in-shell units were fabricated from digital models using a bioprinter equipped with a high-precision plunger dispenser and pressure-based thermoplastic printhead. The printed units were characterized in terms of mass, dimensions, mechanical properties and release performance using paracetamol as a reference tracer. Bacterial viability was evaluated during screening of the formulation components and after each processing step by manual counting of colony-forming units.

Results: A mannitol-based formulation was selected for fabrication of the core, offering a favorable balance of printability, physico-technological properties, release behavior and ability to preserve bacterial viability. Two-layer core-in-shell systems were manufactured via a dual-printing operating mode. The desired in vitro performance was attained, with no release under acidic conditions, a lag phase in pH 6.8 fluid and a subsequent release profile comparable with that generated by the core as such. Viability studies demonstrated that compounding, core printing, shell deposition and drying did not adversely affect L. paracasei survival.

Conclusions: 3D bioprinting was proved to be a versatile technique for the manufacturing of oral colon delivery systems containing probiotics or live biotherapeutics.

Introduction

The gut microbiota constitutes a complex, dynamic ecosystem of bacteria, fungi, viruses, and protozoa that interacts mutually with the host [1]. The relevant composition varies considerably along the gastrointestinal tract, with the colon representing the most densely populated and metabolically active region. Imbalance in this community, commonly termed dysbiosis, has been associated with the pathogenesis of several chronic conditions, not limited to the gastrointestinal tract [2,3]. Particularly, colonic pathologies affected by dysbiosis encompass inflammatory bowel disease (IBD) such as Crohn’s disease and ulcerative colitis [4]. The global incidence of IBD and other intestinal disorders, including colorectal cancer (CRC), irritable bowel syndrome (IBS) and diverticular disease, continues to rise, fueled by Westernized diets, aging populations and sedentary lifestyles [5,6,7]. It has also been reported that chronic inflammation markedly elevates CRC risk [8]. Hence, there is growing interest in therapeutic strategies that may restore microbial balance. Probiotics, defined by the FAO/WHO (2001) as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”, are a cornerstone of such strategies [9]. Lactobacillus, Bifidobacterium, and Streptococcus are frequently used genera alongside yeasts such as Saccharomyces boulardii. These microorganisms exert several beneficial effects, mainly consisting of immune modulation, enhancement of epithelial barrier integrity, production of antimicrobial compounds and competitive exclusion of pathogens [10]. However, orally administered bacteria are required to reach the large bowel in a viable form. A first, major challenge lies in preserving viability throughout production and storage. Indeed, this may be impaired in solid dosage forms such as tablets, where mechanical compaction forces and locally elevated temperatures, beyond oxygen and moisture, can reduce survival rates [11,12]. Certain binders, lubricants and disintegrants may interact adversely with probiotic cells [13]. Coating processes aimed at modified release could also involve threatening conditions. Large-scale production would bring about further stability issues related to supply chain and long-term storage [14]. Substantial declines in viable counts are often observed even during refrigerated storage, with numbers dropping below the therapeutic threshold prior to expiry [15,16,17].

Following intake, bacteria are exposed to the acidic gastric fluid, bile salts and digestive enzymes in the upper gastrointestinal tract, which may seriously harm their viability and colonization potential [18]. Moreover, conventional “one-size-fits-all” products would overlook intra- and inter-subject differences in gut microbiota composition, limiting implementation of precision bacteriotherapy or personalized supplementation treatments that meet the highly diverse needs of each individual patient or consumer [19]. Therefore, for tailored and effective probiotic administration, the carrier dosage form should not only maintain cell viability throughout all manufacturing, storage and usage phases, but also release its bioactive payload selectively into the colon and be amenable to customization in terms of type and dose of bacteria delivered.

Three-dimensional (3D) printing, encompassing a range of additive manufacturing techniques, has recently been leveraged for the fabrication of advanced oral colon delivery systems, mainly based on pH-dependent targeting, containing mesalazine, budesonide, camptothecin, oxalilplatin or different model drugs [20,21,22,23,24,25,26]. To this end, fused deposition modeling (FDM) has primarily been exploited.

Among 3D printing techniques, bioprinting employs bio-inks that are deposited layer by layer to achieve functional biocompatible structures [27,28,29]. Besides its broad appeal for biomedical uses, it holds potential for novel drug delivery applications. Interestingly, bioprinted probiotic formulations for colonic release have already been described [30,31]. Indeed, such a technique could fulfill the previously discussed requirements, allowing for extemporaneous fabrication of custom-designed formulations, avoiding the high compaction pressures and elevated temperatures of conventional processes, namely tableting and coating, facilitating the use of bacteria-friendly formulations and providing geometries that can help protect bacteria from the environment [32]. Like other 3D printing modes, bioprinting would also enable prototyping for relatively larger-scale manufacturing [33,34]. Reported cases of real-world deployment of 3D printers, also in GMP-compliant configurations, would support the possibility of point-of-care, on-demand manufacturing of personalized dosage forms [35].

Based on these premises, the present study was aimed at developing a 3D-bioprinted oral colon delivery system housing, shielding and conveying live microorganisms for bespoke bacteriotherapy applications. A single-parameter formulation strategy for colonic release was preferred over hybrid ones in light of the simpler composition and design to implement in this study. Particularly, a time-dependent approach was followed, relying on the relatively consistent small intestinal transit time of dosage forms, as supported by broad and diverse datasets [36,37,38]. Accordingly, the delivery system was conceived as a reservoir form, embedding the bacteria within a monolithic solid core. The core was surrounded by a swellable hydrophilic polymer barrier, typically based on cellulose derivatives subject to hydration, erosion and dissolution processes upon contact with intestinal fluids over a programmable timespan. Externally, an enteric layer was needed to preserve the inner compartments during unpredictable gastric residence, thus allowing for site-targeted release. Notably, the two-layer core-in-shell configuration pursued represented a challenging goal to achieve via 3D bioprinting.

Lacticaseibacillus paracasei, formerly Lactobacillus paracasei, was employed to address the formulation and fabrication of the dosage form and assess the relevant impact on cell viability. L. paracasei is a Gram-positive member of the lactic acid bacteria group, isolated from the gastrointestinal tract of humans and animals, fermented foods and plant-based substrates [39]. The optimal growth temperature is in the 10–37 °C range, with viability declining sharply beyond it and ceasing above 40 °C. Storage at a refrigerated temperature (4 °C) enhances survival rates [40]. While such overall features posed processing and stability challenges, formulation development could still have been undertaken in the experimental setting where the study was conducted, making the bacterium an appropriate model for this preliminary investigation. Over the past decade, L. paracasei has garnered attention due to a broad spectrum of health-promoting activities [41,42]. Its use has also been explored in the management of gastrointestinal disorders including diarrhea [43], IBS [44] and IBD [45]. A long-standing safety profile and role in maintaining mucosal and microbial homeostasis within the host microbiota have been documented [46].

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Materials

Crospovidone (Kollidon® CL, BASF Italia S.p.A., Cesano Maderno, Italy); hydroxypropyl cellulose (HPC, Klucel™ LF, Ashland, Milan, Italy); hydroxypropyl methylcellulose (HPMC) grades Methocel® E5 Premium LV and Methocel® E50 Premium LV (Colorcon, Dartford, UK); hydroxypropyl methylcellulose acetate succinate (HPMCAS, AQOAT®, Shin-Etsu Chemical Co., Tokyo, Japan); lactose (Lactochem® Crystals, DFE Pharma, Goch, Germany); mannitol grades Mannogem® Powder and Mannogem® XL (SPI Pharma, Septèmes-les-Vallons, France); colloidal silicon dioxide (Aerosil® 200, Evonik Italia S.p.A., Pandino, Italy); paracetamol fine powder (Rhodia, Bollate, Italy); poly(vinyl acetate) and povidone blend (Kollidon® SR, BASF Italia S.p.A.); polyethylene glycol 400 and 1500 (PEG 400 and PEG 1500, Clariant Masterbatches, Milan, Italy); sodium lauryl sulfate (Evonik Italia S.p.A.); sodium starch glycolate (Explotab® CLV, JRS Rettenmaier Italia, Castenedolo, Italy); triethyl citrate (TEC, Sigma-Aldrich, Milan, Italy).

Buscarini, A.; Moutaharrik, S.; Meroni, G.; Cerea, M.; Coldani, M.E.; Foppoli, A.; Palugan, L.; Gazzaniga, A.; Martino, P.A.; Maroni, A. 3D Bioprinting of an Oral Colon Delivery System for Precision Bacteriotherapy. Pharmaceutics 2026, 18, 735. https://doi.org/10.3390/pharmaceutics18060735