3D Printed pH-responsive Colonic Capsules for the Delivery of Live Aqueous Bacterial Suspensions
Abstract
Delivering live bacterial therapeutics to the colon orally is challenging due to the harsh gastrointestinal (GI) conditions and/or the manufacturing processes involved in the production of dry formulations, which can drastically decrease cell viability. In previous work, we evaluated the performance of a 3D printed pH-responsive capsule capable of encapsulating aqueous cargos.
Highlights
- Aqueous live bacterial suspensions die under simulated GI conditions without protection.
- 3D printed capsules release bacteria in simulated colonic conditions (>75% viability)
- TaqMan assays detect strains in feces; microbiome screened prior to delivery.
- 3D printed capsules open in vivo in beagle dogs.
- DNA levels tracked post-delivery of liquid or encapsulated bacteria in beagle dogs.
We herein evaluate its ability to encapsulate live bacterial suspensions with limited processing steps. The capsules maintained their integrity in conditions simulating the upper GI tract (stomach and proximal intestine) and only released their contents in the environment of the lower intestine, i.e. ileum and colon. The mean viability of individual or mixed selected strains remained above 75% during a full simulated GI transit to the colon. In beagle dogs, genomic DNA of 2 out of the 3 delivered strains was detected in the feces, and DNA copy levels did not differ between the capsules and the control suspension of non-encapsulated bacteria.
These results could be attributed to the differing physiological conditions of fasted beagle dogs vs. the simulated environments, or possibly to a non-optimal assessment of bacterial colonization. A follow-up study after capsule treatment, incorporating sampling from various colonic tissues and fluids, could provide key insights into bacterial colonization.
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Materials
Sodium chloride (NaCl) ≥ 99.5%, polysorbate 80 (P80, Tween® 80), isopropyl alcohol (IPA) ≥99.8% ACS reagent, ethanol, dichloromethane (DCM), D-(+)-trehalose dihydrate from starch ≥99.0%, poly(ε−caprolactone) diol 2 kDa (PCL), sodium sulfate (NaSO4) anhydrous ≥99.0%, (+)-α-tocopherol (vitamin E), phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (BAPO), 1-(phenyldiazenyl)naphthalen-2-ol (Sudan I), 1,6-hexanediol dimethacrylate (HDDMA), BAs (sodium cholate and sodium deoxycholate), sodium hydroxide (NaOH), potassium phosphate monobasic (KH2PO4), and L-cysteine hydrochloride, and Anaerotest were purchased from Sigma Aldrich. Celite was obtained from Roth AG. Triethylamine (TEA) 99.0%, methacryloyl chloride 97.0%, and tetrahydrofuran (THF) were acquired from Acros Organics. Enterococcus faecium ATCC® 8459™, Lactobacillus rhamnosus ATCC® 7469™, Bacillus subtilis ATCC® 6051™, and Bifidobacterium breve ATCC® 15700™ Kwik sticks were procured from Chemie Braunschweig. (MRS) broth, MRS agar, TS agar and TS broth, lysogeny broth (LB) were sourced from Merck Millipore.
Hydrochloric acid (HCl) 37% and glycerol 99+% were purchased from VWR chemicals. BD Difco™ agar bacteriological and LIVE/DEAD™ BacLight™ bacterial viability kit for microscopy & quantitative assays containing green fluorescent nucleic acid Stain SYTO9 (3.34 mM) and propidium iodide (20 mM), ultrapure DNase/RNase free water, TAE buffer (Tris(2M)-acetate(1M)–EDTA (0.05 M)) (50X), and TaqMan™ microbe detection kit and TaqPath BactoPure microbial detection master mix containing (Hot-start AmpliTaq fast DNA polymerase, heat-labile uracil-DNA glycosylase (UNG), dNTPs (with dUTP) and optimized buffer and ROX) and Oxoid™ AnaeroGen™ were obtained from Thermo Fisher Scientific. Chloroform-d (CDCl3) was purchased from Apollo Scientific. Lecithin from soybean was ordered from AppliChem. Eudragit® S100 was a gift from Evonik. The Profill poly(vinyl alcohol) (PVAI) easy-print filament was purchased from 3D-Printerstore. Nano pure Milli-Q water was deionized and purified with a Synergy UV water purification system of Merck Millipore. Primers and TaqMan probes were ordered from Microsynth. Colony PCR master mix and O’Gene Ruler DNA Ladder Mix Ready-to-Use 100-10 000 bp were procured from Fischer Scientific. qPCR plates and micro-adhesive plate sealing films were obtained from 4titude. DNeasy 96 PowerSoil Pro Kit and DNeasy® UltraClean® microbial kit were obtained from Qiagen. GoTaq® G2 Master Mix was obtained from Promega. All chemicals were used without further purification.
Fatma Abdi, Marina Green Buzhor, Nadia Zellweger, Rita Maria Kenaan El Rahbani, Daniel Gao, Simon-Pierre Gravel, Michael Burger, Davide Brambilla, Jean-Christophe Leroux, 3D Printed pH-responsive Colonic Capsules for the Delivery of Live Aqueous Bacterial Suspensions, Journal of Drug Delivery Science and Technology, 2024, 106444, ISSN 1773-2247, https://doi.org/10.1016/j.jddst.2024.106444.
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