Bottom-Up Fabrication of Multilayer Enteric Devices for the Oral Delivery of Peptides.

PURPOSE: To develop a planar, asymmetric, micro-scale oral drug delivery vehicle by i) fabricating microdevice bodies with enteric materials, ii) efficiently and stably loading sensitive drug molecules, and iii) capping microdevices for controlled drug release. METHODS: Picoliter-volume inkjet printing was used to fabricate microdevices through additive manufacturing via drop-by-drop deposition of enteric polymer materials. Microdevice bodies with reservoirs are fabricated through deposition of an enteric polymer, Eudragit FS 30 D. A model API, insulin, was loaded into each microdevice and retained its stability during printing and release. Eudragit L 100 and/or S 100 were used to cap microdevices and control the kinetics of insulin release in simulated intestinal conditions. RESULTS: Microdevice morphologies and size can be tuned on the fly based on printing parameters to span from the microscale to the mesoscale. Insulin retained its stability throughout device fabrication and during in vitro release in simulated intestinal conditions. Insulin release kinetics, from burst release to no release, can be tailored by controlling the blend of the Eudragit capping material. CONCLUSION: This approach represents a uniquely scalable and flexible strategy for microdevice fabrication that overcomes limitations in loading sensitive biologics and in the tuneability of device geometries that are inherent to traditional microfabrication strategies.

Nanotopography Enhances Dynamic Remodeling of Tight Junction Proteins through Cytosolic Liquid Complexes.

Nanotopographic materials provide special biophysical stimuli that can regulate epithelial tight junctions and their barrier function. Through the use of total internal reflection fluorescence microscopy of live cells, we demonstrated that contact of synthetic surfaces with defined nanotopography at the apical surface of epithelial monolayers increased paracellular permeability of macromolecules. To monitor changes in tight junction morphology in live cells, we fluorescently tagged the scaffold protein zonula occludens-1 (ZO-1) through CRISPR/Cas9-based gene editing to enable live cell tracking of ZO-1 expressed at physiologic levels. Contact between cells and nanostructured surfaces destabilized junction-associated ZO-1 and promoted its arrangement into highly dynamic liquid cytosolic complexes with a 1-5 µm diameter. Junction-associated ZO-1 rapidly remodeled, and we observed the direct transformation of cytosolic complexes into junction-like structures. Claudin-family tight junction transmembrane proteins and F-actin also were associated with these ZO-1 containing cytosolic complexes. These data suggest that these cytosolic structures are important intermediates formed in response to nanotopographic cues that facilitate rapid tight junction remodeling in order to regulate paracellular permeability.

Picoliter-volume inkjet printing into planar microdevice reservoirs for low-waste, high-capacity drug loading.

Oral delivery of therapeutics is the preferred route for systemic drug administration due to ease of access and improved patient compliance. However, many therapeutics suffer from low oral bioavailability due to low pH and enzymatic conditions, poor cellular permeability, and low residence time. Microfabrication techniques have been used to create planar, asymmetric microdevices for oral drug delivery to address these limitations. The geometry of these microdevices facilitates prolonged drug exposure with unidirectional release of drug toward gastrointestinal epithelium. While these devices have significantly enhanced drug permeability in vitro and in vivo, loading drug into the micron-scale reservoirs of the devices in a low-waste, high-capacity manner remains challenging. Here, we use picoliter-volume inkjet printing to load topotecan and insulin into planar microdevices efficiently. Following a simple surface functionalization step, drug solution can be spotted into the microdevice reservoir. We show that relatively high capacities of both topotecan and insulin can be loaded into microdevices in a rapid, automated process with little to no drug waste.

Fabrication of Sealed Nanostraw Microdevices for Oral Drug Delivery.

The oral route is preferred for systemic drug administration and provides direct access to diseased tissue of the gastrointestinal (GI) tract. However, many drugs have poor absorption upon oral administration due to damaging enzymatic and pH conditions, mucus and cellular permeation barriers, and limited time for drug dissolution. To overcome these limitations and enhance oral drug absorption, micron-scale devices with planar, asymmetric geometries, termed microdevices, have been designed to adhere to the lining of the GI tract and release drug at high concentrations directly toward GI epithelium. Here we seal microdevices with nanostraw membranes-porous nanostructured biomolecule delivery substrates-to enhance the properties of these devices. We demonstrate that the nanostraws facilitate facile drug loading and tunable drug release, limit the influx of external molecules into the sealed drug reservoir, and increase the adhesion of devices to epithelial tissue. These findings highlight the potential of nanostraw microdevices to enhance the oral absorption of a wide range of therapeutics by binding to the lining of the GI tract, providing prolonged and proximal drug release, and reducing the exposure of their payload to drug-degrading biomolecules.

Nanotopography-Induced Structural Anisotropy and Sarcomere Development in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells.

Understanding the phenotypic development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is a prerequisite to advancing regenerative cardiac therapy, disease modeling, and drug screening applications. Lack of consistent hiPSC-CM in vitro data can be largely attributed to the inability of conventional culture methods to mimic the structural, biochemical, and mechanical aspects of the myocardial niche accurately. Here, we present a nanogrid culture array comprised of nanogrooved topographies, with groove widths ranging from 350 to 2000 nm, to study the effect of different nanoscale structures on the structural development of hiPSC-CMs in vitro. Nanotopographies were designed to have a biomimetic interface, based on observations of the oriented myocardial extracellular matrix (ECM) fibers found in vivo. Nanotopographic substrates were integrated with a self-assembling chimeric peptide containing the Arg-Gly-Asp (RGD) cell adhesion motif. Using this platform, cell adhesion to peptide-coated substrates was found to be comparable to that of conventional fibronectin-coated surfaces. Cardiomyocyte organization and structural development were found to be dependent on the nanotopographical feature size in a biphasic manner, with improved development achieved on grooves in the 700-1000 nm range. These findings highlight the capability of surface-functionalized, bioinspired substrates to influence cardiomyocyte development, and the capacity for such platforms to serve as a versatile assay for investigating the role of topographical guidance cues on cell behavior. Such substrates could potentially create more physiologically relevant in vitro cardiac tissues for future drug screening and disease modeling studies.

Micro/nanofabricated platforms for oral drug delivery.

The oral route of drug administration is most preferred due to its ease of use, low cost, and high patient compliance. However, the oral uptake of many small molecule drugs and biotherapeutics is limited by various physiological barriers, and, as a result, drugs suffer from issues with low solubility, low permeability, and degradation following oral administration. The flexibility of micro- and nanofabrication techniques has been used to create drug delivery platforms designed to address these barriers to oral drug uptake. Specifically, micro/nanofabricated devices have been designed with planar, asymmetric geometries to promote device adhesion and unidirectional drug release toward epithelial tissue, thereby prolonging drug exposure and increasing drug permeation. Furthermore, surface functionalization, nanotopography, responsive drug release, motion-based responses, and permeation enhancers have been incorporated into such platforms to further enhance drug uptake. This review will outline the application of micro/nanotechnology to specifically address the physiological barriers to oral drug delivery and highlight technologies that may be incorporated into these oral drug delivery systems to further enhance drug uptake.

Enhanced chondrogenic differentiation of dental pulp stem cells using nanopatterned PEG-GelMA-HA hydrogels.

We have examined the effects of surface nanotopography and hyaluronic acid (HA) on in vitro chondrogenesis of dental pulp stem cells (DPSCs). Ultraviolet-assisted capillary force lithography was employed to fabricate well-defined nanostructured scaffolds of composite PEG-GelMA-HA hydrogels that consist of poly(ethylene glycol) dimethacrylate (PEGDMA), methacrylated gelatin (GelMA), and HA. Using this microengineered platform, we first demonstrated that DPSCs formed three-dimensional spheroids, which provide an appropriate environment for in vitro chondrogenic differentiation. We also found that DPSCs cultured on nanopatterned PEG-GelMA-HA scaffolds showed a significant upregulation of the chondrogenic gene markers (Sox9, Alkaline phosphatase, Aggrecan, Procollagen type II, and Procollagen type X), while downregulating the pluripotent stem cell gene, Nanog, and epithelial-mesenchymal genes (Twist, Snail, Slug) compared with tissue culture polystyrene-cultured DPSCs. Immunocytochemistry showed more extensive deposition of collagen type II in DPSCs cultured on the nanopatterned PEG-GelMA-HA scaffolds. These findings suggest that nanotopography and HA provide important cues for promoting chondrogenic differentiation of DPSCs.