Layer-by-Layer Encapsulation of Probiotics for Delivery to the Microbiome

Recent discoveries in biology and microbiology have highlighted the importance of the gastrointestinal (GI) microbiome in regulating human health and disease. Thus, the delivery of probiotics to influence and modulate microbiome compositions can potentially impact the treatment of a number of human diseases. Unfortunately, biological challenges encountered during oral delivery have limited the translation of many probiotic-delivering technologies. Here, we report a layer-by-layer (LbL) method for the encapsulation of probiotics to directly address these challenges by protecting probiotics from GI tract insults while facilitating both mucoadhesion and direct growth on intestinal surfaces

Rapid, Single-Cell Analysis and Discovery of Vectored mRNA Transfection In Vivo with a loxP-Flanked tdTomato Reporter Mouse

mRNA therapeutics hold promise for the treatment of diseases requiring intracellular protein expression and for use in genome editing systems, but mRNA must transfect the desired tissue and cell type to be efficacious. Nanoparticle vectors that deliver the mRNA are often evaluated using mRNA encoding for reporter genes such as firefly luciferase (FLuc); however, single-cell resolution of mRNA expression cannot generally be achieved with FLuc, and, thus, the transfected cell populations cannot be determined without additional steps or experiments. To more rapidly identify which types of cells an mRNA formulation transfects in vivo, we describe a Cre recombinase (Cre)-based system that permanently expresses fluorescent tdTomato protein in transfected cells of genetically modified mice. Following in vivo application of vectored Cre mRNA, it is possible to visualize successfully transfected cells via Cre-mediated tdTomato expression in bulk tissues and with single-cell resolution. Using this system, we identify previously unknown transfected cell types of an existing mRNA delivery vehicle in vivo and also develop a new mRNA formulation capable of transfecting lung endothelial cells. Importantly, the same formulations with mRNA encoding for fluorescent protein delivered to wild-type mice did not produce sufficient signal for any visualization in vivo, demonstrating the significantly improved sensitivity of our Cre-based system. We believe that the system described here may facilitate the identification and characterization of mRNA delivery vectors to new tissues and cell types

Fabrication of fillable microparticles and other complex 3D microstructures

Three-dimensional (3D) microstructures created by microfabrication and additive manufacturing have demonstrated value across a number of fields, ranging from biomedicine to microelectronics. However, the techniques used to create these devices each have their own characteristic set of advantages and limitations with regards to resolution, material compatibility, and geometrical constraints that determine the types of microstructures that can be formed. We describe a microfabrication method, termed StampEd Assembly of polymer Layers (SEAL), and create injectable pulsatile drug-delivery microparticles, pH sensors, and 3D microfluidic devices that we could not produce using traditional 3D printing. SEAL allows us to generate microstructures with complex geometry at high resolution, produce fully enclosed internal cavities containing a solid or liquid, and use potentially any thermoplastic material without processing additives

Designing Bioactive Delivery Systems for Tissue Regeneration

The direct infusion of macromolecules into defect sites generally does not impart adequate physiological responses. Without the protection of delivery systems, inductive molecules may likely redistribute away from their desired locale and are vulnerable to degradation. In order to achieve efficacy, large doses supplied at interval time periods are necessary, often at great expense and ensuing detrimental side effects. The selection of a delivery system plays an important role in the rate of re-growth and functionality of regenerating tissue: not only do the release kinetics of inductive molecules and their consequent bioactivities need to be considered, but also how the delivery system interacts and integrates with its surrounding host environment. In the current review, we describe the means of release of macromolecules from hydrogels, polymeric microspheres, and porous scaffolds along with the selection and utilization of bioactive delivery systems in a variety of tissue-engineering strategies

A heat-stable microparticle platform for oral micronutrient delivery

Micronutrient deficiencies affect up to 2 billion people and are the leading cause of cognitive and physical disorders in the developing world. Food fortification is effective in treating micronutrient deficiencies; however, its global implementation has been limited by technical challenges in maintaining micronutrient stability during cooking and storage. We hypothesized that polymer-based encapsulation could address this and facilitate micronutrient absorption. We identified poly(butylmethacrylate-co-(2-dimethylaminoethyl)methacrylate-co-methylmethacrylate) (1:2:1) (BMC) as a material with proven safety, offering stability in boiling water, rapid dissolution in gastric acid, and the ability to encapsulate distinct micronutrients. We encapsulated 11 micronutrients (iron; iodine; zinc; and vitamins A, B2, niacin, biotin, folic acid, B12, C, and D) and co-encapsulated up to 4 micronutrients. Encapsulation improved micronutrient stability against heat, light, moisture, and oxidation. Rodent studies confirmed rapid micronutrient release in the stomach and intestinal absorption. Bioavailability of iron from microparticles, compared to free iron, was lower in an initial human study. An organotypic human intestinal model revealed that increased iron loading and decreased polymer content would improve absorption. Using process development approaches capable of kilogram-scale synthesis, we increased iron loading more than 30-fold. Scaled batches tested in a follow-up human study exhibited up to 89% relative iron bioavailability compared to free iron. Collectively, these studies describe a broad approach for clinical translation of a heat-stable ingestible micronutrient delivery platform with the potential to improve micronutrient deficiency in the developing world. These approaches could potentially be applied toward clinical translation of other materials, such as natural polymers, for encapsulation and oral delivery of micronutrients

Methods for Sterilization of Biopolymers for Biomedical Applications

Biopolymers have been found useful in biomedical applications because of their biocompatibility and degradability in the human body. Biopolymers can be formed naturally in living organisms and include polypeptides from proteins, polysaccharides from polymeric carbohydrates and polynucleotides from nucleic acids – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Biopolymers can also be synthesized by using natural biological materials such as starch, sugars, fats, cellulose, and oils. Unsterilized biopolymers can cause severe infections in the human body when they are used for biomedical applications. Hence, biopolymers are required to undergo sterilization, which is a process to inactivate microorganisms including bacteria, spores, fungus, and viruses. The biopolymers that have been sterilized include both the natural and synthetic biodegradable polymers such as chitosan, hyaluronic acid, polylactic acid, poly-L-lactic acid, and poly(lactide-co-glycolide), are being reviewed in this paper. Various sterilization methods that have been applied on biopolymers include steam-autoclaving, dry heat sterilization, irradiation (gamma, X-rays, ultraviolet, and electron beam), chemical treatment (ethylene oxide), gas plasma, and supercritical fluid sterilization, are reviewed

Nanofibrillar Patches of Commensal Skin Bacteria

We demonstrate entrapment of the commensal skin bacteria Staphylococcus epidermidis in mats composed of soft nanotubes made by membrane-templated layer-by-layer (LbL) assembly. When cultured in broth, the resulting nanofibrillar patches efficiently delay the escape of bacteria and their planktonic growth, while displaying high steady-state metabolic activity. Additionally, the material properties and metabolic activity can be further tuned by postprocessing the patches with additional polysaccharide LbL layers. These patches offer a promising methodology for the fabrication of bacterial skin dressings for the treatment of skin dysbiosis while preventing adverse effects due to bacterial proliferation