Local Delivery of Streptomycin in Microcontainers Facilitates Colonization of Streptomycin-Resistant Escherichia coli in the Rat Colon.

Oral antibiotic treatment is often applied in animal studies in order to allow establishment of an introduced antibiotic-resistant bacterium in the gut. Here, we compared the application of streptomycin dosed orally in microcontainers to dosage through drinking water. The selective effect on a resistant bacterial strain, as well as the effects on fecal, luminal, and mucosal microbiota composition, were investigated. Three groups of rats (n = 10 per group) were orally dosed with microcontainers daily for 3 days. One of these groups (STR-M) received streptomycin-loaded microcontainers designed for release in the distal ileum, while the other two groups (controls [CTR] and STR-W) received empty microcontainers. The STR-W group was additionally dosed with streptomycin through the drinking water. A streptomycin-resistant Escherichia coli strain was orally inoculated into all animals. Three days after inoculation, the resistant E. coli was found only in the cecum and colon of animals receiving streptomycin in microcontainers but in all intestinal compartments of animals receiving streptomycin in the drinking water. 16S rRNA amplicon sequencing revealed significant changes in the fecal microbiota of both groups of streptomycin-treated animals. Investigation of the inner colonic mucus layer by confocal laser scanning microscopy and laser capture microdissection revealed no significant effect of streptomycin treatment on the mucus-inhabiting microbiota or on E. coli encroachment into the inner mucus. Streptomycin-loaded microcontainers thus enhanced proliferation of an introduced streptomycin-resistant E. coli in the cecum and colon without affecting the small intestine environment. While improvements of the drug delivery system are needed to facilitate optimal local concentration and release of streptomycin, the application of microcontainers provides new prospects for antibiotic treatment. IMPORTANCE Delivery of antibiotics in microcontainer devices designed for release at specific sites of the gut represents a novel approach which might reduce the amount of antibiotic needed to obtain a local selective effect. We propose that the application of microcontainers may have the potential to open novel opportunities for antibiotic treatment of humans and animals with fewer side effects on nontarget bacterial populations. In the current study, we therefore elucidated the effects of streptomycin, delivered in microcontainers coated with pH-sensitive lids, on the selective effect on a resistant bacterium, as well as on the surrounding intestinal microbiota in rats.

Development and characterization of a PDMS-based masking method for microfabricated Oral drug delivery devices.

With the growing popularity and application of microfabricated devices in oral drug delivery (ODD), masking technologies for drug loading and surface modification become highly relevant. Considering the speed of design and fabrication processes and the necessity for continuous alterations of e.g. the shape and sizes of the devices during the optimization process, there is a need for adaptable, precise and low-cost masking techniques. Here, a novel method is presented for masking ODD microdevices, namely microcontainers, using the physical characteristics of polydimethylsiloxane (PDMS). When compared to a rigid microfabricated shadow mask, used for filling drugs in microcontainers, the PDMS masking technique allows more facile and precise loading of higher quantities of an active compound, without the need of alignment. The method provides flexibility and is adjustable to devices fabricated from different materials with various geometries, topologies and dimensions. This user-friendly flexible masking method overcomes the limitations of other masking techniques and is certainly not limited to ODD and is recommended for a wide range of microdevices.

Engineered substrates incapable of induction of chondrogenic differentiation compared to the chondrocyte imprinted substrates.

It is well established that surface topography can affect cell functions. However, finding a reproducible and reliable method for regulating stem cell behavior is still under investigation. It has been shown that cell imprinted substrates contain micro- and nanoscale structures of the cell membrane that serve as hierarchical substrates, can successfully alter stem cell fate. This study investigated the effect of the overall cell shape by fabricating silicon wafers containing pit structure in the average size of spherical-like chondrocytes using photolithography technique. We also used chondrocyte cell line (C28/I2) with spindle-like shape to produce cell imprinted substrates. The effect of all substrates on the differentiation of adipose-derived mesenchymal stem cells (ADSCs) has been studied. The AFM and scanning electron microscopy images of the prepared substrates demonstrated that the desired shapes were successfully transferred to the substrates. Differentiation of ADSCs was investigated by immunostaining for mature chondrocyte marker, collagen II, and gene expression of collagen II, Sox9, and aggrecan markers. C28/I2 imprinted substrate could effectively enhanced chondrogenic differentiation compared to regular pit patterns on the wafer. It can be concluded that cell imprinted substrates can induce differentiation signals better than engineered lithographic substrates. The nanostructures on the cell-imprinted patterns play a crucial role in harnessing cell fate. Therefore, the patterns must include the nano-topographies to have reliable and reproducible engineered substrates.

Bioadhesive Tannic-Acid-Functionalized Zein Coating Achieves Engineered Colonic Delivery of IBD Therapeutics via Reservoir Microdevices.

The biggest challenge in oral delivery of anti-inflammatory drugs such as 5-aminosalicylic acid (5-ASA) is to (i) prevent rapid absorption in the small intestine and (ii) achieve localized release at the site of inflammation in the lower gut, i.e., the colon. Here, we present an advanced biopolymeric coating comprising of tannic-acid-functionalized zein protein to provide a sustained, colon-targeted release profile for 5-ASA and enhance the mucoadhesion of the dosage form via a mussel-inspired mechanism. To enable localized delivery and provide high local concentration, 5-ASA is loaded into the microfabricated drug carriers (microcontainers) and sealed with the developed coating. The functionality and drug release profile of the coating are characterized and optimized in vitro, showing great tunability, scalability, and stability toward proteases. Further, ex vivo experiments demonstrate that the tannic acid functionalization can significantly enhance the mucoadhesion of the coating, which is followed up by in vivo investigations on the intestinal retention, and pharmacokinetic evaluation of the 5-ASA delivery system. Results indicate that the developed coating can provide prolonged colonic delivery of 5-ASA. Therefore, the here-developed biodegradable coating can be an eco-friendly substitute to the state-of-the-art commercial counterparts for targeted delivery of 5-ASA and other small molecule drugs.

Cell-imprinted substrates: in search of nanotopographical fingerprints that guide stem cell differentiation.

Cell-imprinted substrates direct stem cell differentiation into various lineages, suggesting the idea of lineage-specific nanotopography. We herein examined the surface topography of five different imprinted cell patterns using AFM imaging and statistical analysis of amplitude, spatial, and hybrid roughness parameters. The results suggest that different cell imprints possess distinguished nanotopographical features.

Design of a self-unfolding delivery concept for oral administration of macromolecules.

Delivering macromolecular drugs, e.g. peptides, to the systemic circulation by oral administration is challenging due to their degradation in the gastrointestinal tract and low transmucosal permeation. In this study, the concept of an oral delivery device utilizing an elastomeric material is presented with the potential of increasing the absorption of peptides, e.g. insulin. Absorption enhancement in the intestine is proposed as a result of self-unfolding of a polydimethylsiloxane foil upon release from enteric coated capsules. A pH-sensitive polymer coating prevents capsule disintegration until arrival in the small intestine where complete unfolding of the elastomeric foil ensures close contact with the intestinal mucosa. Foils with close-packed hexagonal compartments for optimal drug loading are produced by casting against a deep-etched silicon master. Complete unfolding of the foil upon capsule disintegration is verified in vitro and the insulin release profile of the final delivery device confirms insulin protection at gastric pH. In vivo performance is evaluated with the outcome of quantifiable plasma insulin concentrations in all rats receiving duodenal administration of the novel delivery device. By taking advantage of elastomeric material properties for drug delivery, this approach might serve as inspiration for further development of commercially viable biocompatible devices for oral delivery of macromolecules.

Orally ingestible medical devices for gut engineering.

Orally ingestible medical devices provide significant advancement for diagnosis and treatment of gastrointestinal (GI) tract-related conditions. From micro- to macroscale devices, with designs ranging from very simple to complex, these medical devices can be used for site-directed drug delivery in the GI tract, real-time imaging and sensing of gut biomarkers. Equipped with uni-direction release, or self-propulsion, or origami design, these microdevices are breaking the barriers associated with drug delivery, including biologics, across the GI tract. Further, on-board microelectronics allow imaging and sensing of gut tissue and biomarkers, providing a more comprehensive understanding of underlying pathophysiological conditions. We provide an overview of recent advances in orally ingestible medical devices towards drug delivery, imaging and sensing. Challenges associated with gut microenvironment, together with various activation/actuation modalities of medical devices for micromanipulation of the gut are discussed. We have critically examined the relationship between materials-device design-pharmacological responses with respect to existing regulatory guidelines and provided a clear roadmap for the future.

3D Printing of Reservoir Devices for Oral Drug Delivery: From Concept to Functionality through Design Improvement for Enhanced Mucoadhesion.

So far, microdevices for oral drug delivery have been fabricated as square or cylindrical reservoir structures with a localized and unidirectional release. The fabrication is usually carried out using sophisticated and costly microfabrication techniques. Here, 3D printing of microreservoirs on sacrificial substrates is presented. This approach allows the devices to be accurately arranged in predetermined patterns, enabling implementation into batch production schemes in which the fabrication of the devices is linked to processing steps such as automated drug loading and sealing. Moreover, design and 3D printing of alternative geometries of minireservoirs featuring anchor-like surface structures for improved mucoadhesion and intestinal retention is demonstrated. Surface texturing of minireservoirs increases mucoadhesion of the devices up to two-fold compared to a nonstructured control. The structuring also leads to a strong bias in mucoadhesion in different orientations, which can facilitate a correct orientation of the devices and thus lead to unidirectional release of drugs toward the intestinal mucosa for increased drug uptake.

Tenocyte-imprinted substrate: a topography-based inducer for tenogenic differentiation in adipose tissue-derived mesenchymal stem cells.

Tendon tissue engineering based on stem cell differentiation has attracted a great deal of attention in recent years. Previous studies have examined the effect of cell-imprinted polydimethylsiloxane (PDMS) substrate on induction differentiation in stem cells. In this study, we used tenocyte morphology as a positive mold to create a tenocyte-imprinted substrate on PDMS. The morphology and topography of this tenocyte replica on PDMS was evaluated with scanning electron microscopy (SEM) and atomic force microscopy. The tenogenic differentiation induction capacity of the tenocyte replica in adipose tissue-derived mesenchymal stem cells (ADSCs) was then investigated and compared with other groups, including tissue replica (which was produced similarly to the tenocyte replica and was evaluated by SEM), decellularized tendon, and bone morphogenic protein (BMP)-12, as other potential inducers. This comparison gives us an estimate of the ability of tenocyte-imprinted PDMS (called cell replica in the present study) to induce differentiation compared to other inducers. For this reason, ADSCs were divided into five groups, including control, cell replica, tissue replica, decellularized tendon and BMP-12. ADSCs were seeded on each group separately and investigated by the real-time reverse transcription polymerase chain reaction (RT-PCR) technique after seven and 14 days. Our results showed that in spite of the higher effect of the growth factor on tenogenic differentiation, the cell replica can also induce tenocyte marker expression (scleraxis and tenomodulin) in ADSCs. Moreover, the tenogenic differentiation induction capacity of the cell replica was greater than tissue replica. Immunocytochemistry analysis revealed that ADSCs seeding on the cell replica for 14 days led to scleraxis and tenomodulin expression at the protein level. In addition, immunohistochemistry indicated that contrary to the promising results in vitro, there was little difference between ADSCs cultured on tenocyte-imprinted PDMS and untreated ADSCs. The results of such studies could lead to the production of inexpensive cell culture plates or biomaterials that can induce differentiation in stem cells without growth factors or other supplements.

An engineered cell-imprinted substrate directs osteogenic differentiation in stem cells.

A cell-imprinted poly(dimethylsiloxane)/hydroxyapatite nanocomposite substrate was fabricated to engage topographical, mechanical, and chemical signals to stimulate and boost stem cell osteogenic differentiation. The physicochemical properties of the fabricated substrates, with nanoscale resolution of osteoblast morphology, were probed using a wide range of techniques including scanning electron microscopy, atomic force microscopy, dynamic mechanical thermal analysis, and water contact angle measurements. The osteogenic differentiation capacity of the cultured stem cells on these substrates was probed by alizarin red staining, ALP activity, osteocalcin measurements, and gene expression analysis. The outcomes revealed that the concurrent roles of the surface patterns and viscoelastic properties of the substrate provide the capability of directing stem cell differentiation toward osteogenic phenotypes. Besides the physical and mechanical effects, we found that the chemical signaling of osteoinductive hydroxyapatite nanoparticles, embedded in the nanocomposite substrates, could further improve and optimize stem cell osteogenic differentiation.