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Periodontitis is one of the most common chronic inflammatory diseases worldwide [1]. Recently, the overgrowth of human herpes viruses (HHVs), including herpes simplex virus type 1 (HSV-1), has been suggested as a possible trigger for oral dysbiosis resulting in the progression of periodontitis [2]. Thus, there is a demand for in vitro platforms to facilitate studies of the interactions between oral cells (e.g. epithelial cells and fibroblasts) and HHVs in conditions mimicking the oral cavity to enable a better understanding of fundamental biological processes and evaluate new treatment strategies. Traditionally, viral infections are studied in cells cultured in static conditions, e.g. well plates. However, different methods to study cells in perfusion have received increasing attention [3, 4, 5]. A recently established methodology to study cell growth under perfusion in vitro is based on the Lab-on-a-Disc (LoD) [6] technology. LoD is a disc-shaped microfluidic platform the size of a DVD, where centrifugal forces are used to manipulate fluids through channels and chambers [6]. Compared to conventional perfusion systems, LoD platforms are advantageous since there are no external pumps, valves or tubings. In this work, we aim to establish a physiologically relevant LoD model to study dynamic interactions between cells isolated from the oral cavity, e.g. periodontal ligament (PDL) cells, and HHVs as it may occur in periodontitis.

For patients with ulcerative colitis (UC), administration of the probiotic E. coli Nissle (EcN) holds promise for alleviation of disease symptoms. The mechanisms are unclear, but it has been hypothesised that a capacity of the probiotic to outcompete potentially detrimental UC-associated E. coli strains plays an important role. However, this could previously not be confirmed in a mouse model of competition between EcN and two UC-associated strains, as reported by Petersen et al. 2011. In the present study, we re-evaluated the idea, hypothesising that delivery of EcN by a micro device dosing system (microcontainers), designed for delivery into the intestinal mucus, could support colonisation and confer a competition advantage compared to classical oral dosing. Six groups of mice were pre-colonised with one of two UC-associated E. coli strains followed by oral delivery of EcN, either in capsules containing microcontainers with freeze-dried EcN powder, capsules containing freeze-dried EcN powder, or as a fresh sucrose suspension. Co-colonisation between the probiotic and the disease-associated strains was observed regardless of dosing method, and no competition advantages linked to microcontainer delivery were identified within this setup. Other approaches are thus needed if the competitive capacity of EcN in the gut should be improved.

During the past decades, microdevices have been evaluated as a means to overcome challenges within oral drug delivery, thus improving bioavailability. Fabrication of microdevices is often limited to planar or simple 3D designs. Therefore, this work explores how microscale stereolithography 3D printing can be used to fabricate radiopaque microcontainers with enhanced mucoadhesive geometries, which can enhance bioavailability by increasing gastrointestinal retention. Ex vivo force measurements suggest increased mucoadhesion of microcontainers with adhering features, such as pillars and arrows, compared to a neutral design. In vivo studies, utilizing planar x-ray imaging, show time dependent gastrointestinal location of microcontainers, whereas computed tomography scanning and cryogenic scanning electron microscopy reveal information about their spatial dynamics and mucosal interactions. For the first time, the effect of 3D microdevice modifications on gastrointestinal retention is traced in vivo, and the applied methods provide a much-needed approach for investigating the impact of device design on gastrointestinal retention. This article is protected by copyright. All rights reserved.

Combination drug therapy is commonly used to treat cancer, diabetes, cardiovascular conditions, and infections. However, these therapies face challenges associated with patient compliance and toxicology. Over the past decades, microdevices have emerged as a promising candidate for oral delivery allowing for targeted drug delivery with a tunable drug release. In the present work, engineered and monodisperse dual-compartment microdevices are developed to achieve a physical separation of two drugs followed by a sequential release in the gastrointestinal tract. As proof-of-concept, the compartments are sealed with two pH-sensitive polymers of different thicknesses to control the sequential release of propranolol and furosemide. In vitro release studies and in vivo absorption studies in rats confirm a sequential drug release from the two compartments. Unlike other proposed approaches, it is highly advantageous that the drugs can be loaded directly as powders, and that their release can be tuned via optimized coatings to achieve the desired release and absorption profiles. Conclusively, this study lays a strong foundation for the future use of microdevices to enable co-delivery of drugs followed by a sequential release in close proximity in the gastrointestinal tract.

In vitro release and dissolution models are widely used in the development phases of oral drug delivery systems to measure how an active pharmaceutical ingredient (API) is released from a dosage form. However, additional requirements for these models arise when evaluating probiotic dosage forms since they are often sensitive to temperature and oxygen levels. As a solution to this, we propose a custom-designed anaerobic in vitro release setup, made mainly by 3D printing and laser cutting, to function together with state-of-the-art pharmaceutical dissolution equipment – in this case, a microDISS Profiler™. The in vitro release model makes it possible to study the release rate of oxygen-sensitive probiotics in simulated intestinal conditions, while ensuring their survival due to the anaerobic conditions. This has not been possible so far since the available in vitro dissolution models have not been compatible with anaerobic conditions. With two different case studies, the developed model combined with a microDISS Profiler™ has proven capable of measuring the release of a probiotic and a small-molecule API from microdevices for oral drug delivery. Further, the model facilitated the survival of anaerobic bacteria present in the release medium.

Microcontainers, which are microfabricated cylindrical devices with a reservoir function, have shown promise as an oral drug delivery system for small molecular drug compounds. However, they have never been evaluated against a relevant control formulation. In the current study, we prepared microcrystalline cellulose (MCC) microspheres as a control for in vitro and in vivo testing of SU-8 microcontainers as an oral drug delivery system. Both dosage forms were loaded with paracetamol and coated with chitosan or polyethylene glycol (PEG) (12 kDa). These coatings were followed by an additional enteric coating of Eudragit® S100. In addition, a control dosage form was coated with Eudragit® alone. The dosage forms were evaluated in vitro, in a physiologically relevant two-step model simulating rat gastrointestinal fluids, and in vivo by oral administration to rats. In vitro, the microcontainers coated with PEG/Eudragit® resulted in a prolonged release of paracetamol compared to the respective microspheres, which was consistent with in vivo observations of a later time (Tmax) for maximum plasma concentration (Cmax) for the microcontainers. For microspheres and microcontainers coated with chitosan/Eudragit®, the time for complete in vitro release of paracetamol was very similar, due to an earlier release from the microcontainers. This trend was supported by very similar Tmax values in vivo. The in vitro in vivo relation was confirmed by a linear regression with R2 = 0.9, when Tmax for each dosage form was plotted as a function of time for 90% paracetamol release in vitro. From the in vivo study, the average plasma concentration of paracetamol 120 min after dosing was significantly higher for microcontainers than for microspheres (0.3 ± 0.1 µg/mL and 0.1 ± <0.1 µg/mL, respectively) – regardless of the coating applied.

Oral administration of probiotics is beneficial for restoring the intestinal microbial balance and for the treatment of gastrointestinal (GI) tract-related disorders. In the current era characterized by the development of next-generation probiotic microorganisms, which are typically less robust toward environmental challenges than the classically applied lactic acid producing probiotics, we anticipate a need for delivery of live organisms directly to the site where they need to colonize. Here, we thus present, for the first time, a proof of concept for using micrometer-sized polymeric containers, for the engineered delivery of probiotics, using spray dried Lactobacillus rhamnosus GG (LGG), as a model probiotic microorganism. To achieve colon-specific delivery, microcontainers are loaded with LGG and sealed with an enzyme-sensitive coating. A genipin-cross-linked chitosan coating is developed that (i) is stable at gastric and intestinal pH; (ii) has tunable swelling; and (iii) is degradable by the colon-specific bacterial enzymes. The chitosan-genipin coated microcontainers are evaluated in vitro, ex vivo, as well as in vivo in a rat model. Our results confirm that the genipin-cross-linked chitosan coating enables an effective local delivery in the cecum and colon without any premature release in the small intestine. Our findings suggest that the integration of modified polysaccharides with ingestible microdevices has great potential for controlled and site-specific delivery of live microorganisms.

An increased interest in colonic drug delivery has led to a higher focus on the design of delivery devices targeting this part of the gastrointestinal tract. Microcontainers have previously facilitated an increase in oral bioavailability of drugs. The surface texture and shape of microcontainers have proven to influence the mucoadhesion ex vivo. In the present work, these findings were further investigated using an in situ closed-loop perfusion technique in the rat colon, which allowed for simultaneous evaluation of mucoadhesion of the microcontainers as well as drug absorption. Cylindrical, triangular and cubic microcontainers, with the same exterior surface area, were evaluated based on in vitro release, in situ mucoadhesion and in situ absorption of amoxicillin. Additionally, the mucoadhesion of empty cylindrical microcontainers with and without pillars on the top surface was investigated. From the microscopy analysis of the colon sections after the in situ study, it was evident that a significantly higher percentage of cubic microcontainers than cylindrical microcontainers adhered to the intestinal mucus. Furthermore, the absorption rate constants and blood samples indicated that amoxicillin in cubic microcontainers was absorbed more readily than when cylindrical or triangular microcontainers were dosed. This could be due to a higher degree of mucoadhesion for these particular microcontainers.

Oral consumption of probiotic microorganisms is thought to provide various beneficial effects to the host. However, probiotics encounter harsh conditions through the gastrointestinal tract, which may decrease the gut colonisation potential. The polymeric micrometer-sized devices called microcontainers serves as vehicles to protect and deliver probiotics at target locations by using specific biodegradable coatings. The microcontainers have previously been shown to be suitable for improving intestinal drug absorption in rats through oral delivery1. In this pilot study, we allocated 18 germ-free mice in 3 groups, which were dosed with capsules either containing L. rhamnosus GG (LGG), microcontainers filled with LGG and coated with Eudragit® L 100-55 or empty microcontainers. We found that delivery of LGG to the intestine of germ-free mice loaded into microcontainers was comparable to delivery of LGG without microcontainers. Since LGG is highly stable in the gastric environment2, high survival of LGG cells even without confinement in microcontainers was expected. However, the microcontainers have the potential to maintain a high cell survival rate, which also applies to bacteria that are more susceptible, and enhance the gut colonisation potential through targeted delivery. Further investigations are currently being conducted to evaluate the colonisation properties and effects of probiotics delivered to the gut using microcontainers.

Objective In the MIMIO project (Microstructures, Microbiota, and Oral delivery), funded by The Novo Nordisk Foundation, we aim to investigate the application of a novel delivery system based on orally administered probiotic-filled microcontainers. Since loading of the microcontainers, including probiotic preparation and storage, represents a critical challenge in this project, systematic viability tests of bacteria were evaluated following various spray drying and storage conditions. Method The Gram-positive Lactobacillus rhamnosus GG (LGG) and Gram-negative Escherichia coli Nissle 1917 were spray dried at different settings using a BÜCHI Mini Spray Dryer, B-290. Additionally, a combination of trehalose and reconstituted skim milk (RSM) powder was added as protectant. Afterwards, the spray dried powder of LGG was stored at different conditions with variations in light exposure and temperature. Results For LGG, spray drying using an outlet temperature of 60°C and 10 w/w % trehalose and 10 w/w % RSM as protectants resulted in a fine powder with high cell viability, which could be stored for a prolonged period of time. However, spray drying of E. coli Nissle resulted in survival of less than 1 % at all tested conditions. Conclusion Spray drying with the addition of trehalose and RSM is a suitable technique to preserve LGG with a high cell viability. However, E. coli Nissle is more vulnerable to stress related to the spray drying procedure, possibly due to the sensitive Gram-negative cell wall structure. Therefore, we suggest using a different preparation method for this bacterium.

Many drug candidates today have a low aqueous solubility and, hence, may show a low oral bioavailability, presenting a major formulation and drug delivery challenge. One way to increase the bioavailability of these drugs is to use a supersaturating drug delivery strategy. The aim of this study was to develop a video-microscopic method, to evaluate the effect of a precipitation inhibitor on supersaturated solutions of the poorly soluble drug tadalafil, using a novel video-microscopic small scale setup. Based on preliminary studies, a degree of supersaturation of 29 was chosen for the supersaturation studies with tadalafil in FaSSIF. Different amounts of hydroxypropyl methyl cellulose (HPMC) were predissolved in FaSSIF to give four different concentrations, and the supersaturated system was then created using a solvent shift method. Precipitation of tadalafil from the supersaturated solutions was monitored by video-microscopy as a function of time. Single-particle analysis was possible using commercially available software; however, to investigate the entire population of precipitating particles (i.e., their number and area covered in the field of view), an image analysis algorithm was developed (multiparticle analysis). The induction time for precipitation of tadalafil in FaSSIF was significantly prolonged by adding 0.01% (w/v) HPMC to FaSSIF, and the maximum inhibition was reached at 0.1% (w/v) HPMC, after which additional HPMC did not further increase the induction time. The single-particle and multiparticle analyses yielded the same ranking of the HPMC concentrations, regarding the inhibitory effect on precipitation. The developed small scale method to assess the effect of precipitation inhibitors can speed up the process of choosing the right precipitation inhibitor and the concentration to be used.