Delivery of Exenatide and Insulin Using Mucoadhesive Intestinal Devices.

A major disadvantage associated with current diabetes therapy is dependence on injectables for long-term disease management. In addition to insulin, incretin hormone replacement therapies including exenatide have added a new class of drugs for Type-2 diabetes. Although efficacious, patient compliance with current diabetic therapy is poor due to requirement of injections, inability to cross the intestinal epithelium and instability in the gastrointestinal tract. Here, we report the efficacy of a mucoadhesive device in providing therapeutic concentrations of insulin and exenatide via oral administration. Devices were prepared with a blend of FDA-approved polymers, carbopol, pectin and sodium carboxymethylcellulose, and were tested for drug carrying capability, in vitro release, Caco-2 permeability, and in vivo efficacy for insulin and exenatide. Results suggested that mucoadhesive devices successfully provided controlled release of FITC-insulin, released significant amounts of drug, while providing noteworthy enhancement of drug transport across Caco-2 monolayers without compromising monolayer integrity. In-vivo administration of the devices provided significant enhancement of drug absorption with 13- and 80-fold enhancement of relative bioavailability for insulin and exenatide compared to intestinal injections with significant increase in half-lives, thus resulting in prolonged blood glucose reduction. This study validates the efficacy of mucoadhesive devices in promoting oral peptide delivery to improve patient compliance and dose adherence.

Mucoadhesive intestinal devices for oral delivery of salmon calcitonin.

One of the major challenges faced by therapeutic polypeptides remains their invasive route of delivery. Oral administration offers a potential alternative to injections; however, this route cannot be currently used for peptides due to their limited stability in the stomach and poor permeation across the intestine. Here, we report mucoadhesive devices for oral delivery that are inspired by the design of transdermal patches and demonstrate their capabilities in vivo for salmon calcitonin (sCT). The mucoadhesive devices were prepared by compressing a polymeric matrix containing carbopol, pectin and sodium carboxymethylcellulose (1:1:2), and were coated on all sides but one with an impermeable and flexible ethyl cellulose (EC) backing layer. Devices were tested for in vitro dissolution, mucoadhesion to intestinal mucosa, enhancement of drug absorption in vitro (Caco-2 monolayer transport) and in vivo in rats. Devices showed steady drug release with ≈75% cumulative drug released in 5h. Devices also demonstrated strong mucoadhesion to porcine small intestine to withstand forces up to 100 times their own weight. sCT-loaded mucoadhesive devices exhibited delivery of sCT across Caco-2 monolayers and across the intestinal epithelium in vivo in rats. A ≈52-fold (pharmacokinetic) and ≈44-fold (pharmacological) enhancement of oral bioavailability was observed with mucoadhesive devices when compared to direct intestinal injections. Oral delivery of devices in enteric coated capsules resulted in significant bioavailability enhancement.

A permeation enhancer for increasing transport of therapeutic macromolecules across the intestine.

Delivery of therapeutic macromolecules is limited by the physiological limitations of the gastrointestinal tract including poor intestinal permeability, low pH and enzymatic activity. Several permeation enhancers have been proposed to enhance intestinal permeability of macromolecules; however their utility is often hindered by toxicity and limited potency. Here, we report on a novel permeation enhancer, Dimethyl palmitoyl ammonio propanesulfonate (PPS), with excellent enhancement potential and minimal toxicity. PPS was tested for dose- and time-dependent cytotoxicity, delivery of two model fluorescent molecules, sulforhodamine-B and FITC-insulin in vitro, and absorption enhancement of salmon calcitonin (sCT) in vivo. Caco-2 studies revealed that PPS is an effective enhancer of macromolecular transport while being minimally toxic. TEER measurements in Caco-2 monolayers confirmed the reversibility of the effect of PPS. Confocal microscopy studies revealed that molecules permeate via both paracellular and transcellular pathways in the presence of PPS. In vivo studies in rats showed that PPS enhanced relative bioavailability of sCT by 45-fold after intestinal administration. Histological studies showed that PPS does not induce damage to the intestine. PPS is an excellent permeation enhancer which provides new opportunities for developing efficacious oral/intestinal delivery systems for therapeutic macromolecules.

Permeation of insulin, calcitonin and exenatide across Caco-2 monolayers: measurement using a rapid, 3-day system.

OBJECTIVES: Caco-2 monolayers are one of the most widely used in vitro models for prediction of intestinal permeability of therapeutic molecules. However, the conventional Caco-2 monolayer model has several drawbacks including labor-intensive culture process, unphysiological growth conditions, lack of reproducibility and limited throughput. Here, we report on the use of 3-day Caco-2 monolayers for assessing permeability of polypeptide drugs. METHODS: The 3-day monolayers were grown in a commercially available transwell set-up, which facilitates rapid development of the Caco-2 monolayers in an intestinal epithelial differentiation mimicking environment. This set-up included use of serum-free medium of defined composition with supplements such as butyric acid, hormones, growth factors, and other metabolites, reported to regulate the differentiation of intestinal epithelial cells in vivo. We measured permeability of 3 different therapeutic polypeptides; insulin, calcitonin, and exenatide across the monolayer. RESULTS: Preliminary validation of the monolayer was carried out by confirming dose-dependent permeation of FITC-insulin and sulforhodamine-B. Transport of insulin, calcitonin, and exenatide measured at different loading concentrations suggests that the permeability values obtained with 3-day cultures resemble more closely the values obtained with ex vivo models compared to permeability values obtained with conventional 21-day cultures. CONCLUSIONS: Short-term 3-day Caco-2 monolayers provide new opportunities for developing reproducible and high-throughput models for screening of therapeutic macromolecules for oral absorption.

Mucociliary clearance of micro- and nanoparticles is independent of size, shape and charge--an ex vivo and in silico approach.

The fate of inhaled particles after deposition onto the pulmonary mucosa is far from being solved, in particular with respect to mucociliary clearance and mucus penetration. Due to the fact that these phenomena govern pulmonary residence time and thus bioavailability, they are highly relevant for any kind of controlled release formulation delivered via that route. This study applies ex vivo and in silico approaches to investigate the dependency of muciliary clearance of micro-, submicrometer and nanoparticles on size, shape, charge and surface chemistry of such particles. In addition, measurement of mucociliary clearance of different particles also provided information about their penetration into mucus. Surprisingly, no significant differences in mucociliary clearance could be found for any type of particle under investigation. As revealed by computational modeling, particle penetration into the mucus gel layer was negligible at least within the time frame allowed by horizontal mucus transport. These data suggest that the observed lack of difference in mucociliary clearance is caused by the lack of immediate penetration of deposited aerosol particles through the mucus blanket.

A reagent to facilitate protein recovery from cells and tissues.

Collection of cytosolic proteins from cells and tissues is the first and essential step in many bioanalytical assays that play a key role in medical applications such as diagnostics, theranostics, and regenerative medicine. Dissolution of cell and tissue constituents without deactivation of their constituents, especially proteins, is a challenging task. Here, we report on a recently identified cell and tissue lysis agent in terms of its ability to solubilize cells and tissues as well as preservation of cellular proteins, particularly enzymes. The lysis agent comprises a mixture of a zwitterionic surfactant, N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (DPS) and a non-ionic surfactant, Brij 30 (B30). Mixtures of DPS and B30 successfully solubilized keratinocytes and human vascular endothelial cells in vitro while preserving detectable quantities of cellular enzymes including glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase for at least 4 h. Mixtures of DPS and B30 were also effective in solubilizing tissues, especially tough tissues such as skin in vitro. Collectively, the mixture of DPS/B30 was effective in solubilizing cells and tissues while preserving its constituent proteins, which opens up its applications for use in studying the effect of environmental factors on tissue proteomics. As an example, the ability of DPS/B30 to detect alterations in skin proteins in response to UV exposure was assessed. These studies revealed that UV exposure induce upregulation of a number of inflammatory, apoptotic and stress-activated proteins as well as downregulation of cell cycle progression proteins.

Polymer nanoneedle-mediated intracellular drug delivery.

Delivery of drugs into the cellular cytoplasm of target cells represents a major hurdle in treating various diseases. This challenge can be addressed by encapsulation of drugs onto or within nanoparticles, which can then be targeted to diseased cells. Here, needle-shaped particles are shown to exhibit substantially higher cytoplasmic delivery of drugs such as siRNA compared to their spherical counterparts. Furthermore, these needles are designed to lose their sharp tips over time and can render themselves ineffective over time, thereby offering control over their duration of activity and toxicity. Such polymer nanoneedles open new avenues for delivering drug molecules directly into the cytoplasm with low toxicity.

Adaptive micro and nanoparticles: temporal control over carrier properties to facilitate drug delivery.

Recent studies have led to significant advances in understanding the impact of key drug carrier properties such as size, surface chemistry and shape on their performance. Converting this knowledge into improved therapeutic outcomes, however, has proved challenging. This owes to the fact that successful drug delivery carriers have to navigate through multiple physiological hurdles including reticuloendothelial system (RES) clearance, target accumulation, intracellular uptake and endosomal escape. Each of these processes may require unique, and often conflicting, design parameters, thus making it difficult to choose a design that addresses all these hurdles. This challenge can be addressed by designing carriers whose properties can be changed in time so as to successfully navigate them through various biological hurdles. Several carriers have been reported that implement this strategy. This review will discuss the current status and future prospects of this emerging field of "adaptive micro and nanoparticles".

Needle-shaped polymeric particles induce transient disruption of cell membranes.

Nano- and microparticles of various shapes have recently been introduced for various drug-delivery applications. Shape of particles has been shown to have an impact on various processes including circulation, vascular adhesion and phagocytosis. Here, we assess the role of particle geometry and surface chemistry in their interactions with cell membranes. Using representative particles of different shape (spheres, elongated and flat particles), size (500 nm-1 microm) and surface chemistry (positively and negatively charged), we evaluated the response of endothelial cells to particles. While spherical and elliptical disc-shaped particles did not have an impact on cell spreading and motility, needle-shaped particles induced significant changes in the same. Further studies revealed that needle-shaped particles induced disruption of cell membranes as indicated by the release of lactate dehydrogenase and uptake of extracellular calcein. The effect of needle-shaped particles on cells was transient and was reversed over a time period of 1-48 h depending on particle parameters.

Flow and adhesion of drug carriers in blood vessels depend on their shape: a study using model synthetic microvascular networks.

Development of novel carriers and optimization of their design parameters has led to significant advances in the field of targeted drug delivery. Since carrier shape has recently been recognized as an important design parameter for drug delivery, we sought to investigate how carrier shape influences their flow in the vasculature and their ability to target the diseased site. Idealized synthetic microvascular networks (SMNs) were used for this purpose since they closely mimic key physical aspects of real vasculature and at the same time offer practical advantages in terms of ease of use and direct observation of particle flow. The attachment propensities of surface functionalized spheres, elliptical/circular disks and rods with dimensions ranging from 1microm to 20microm were compared by flowing them through bifurcating SMNs. Particles of different geometries exhibited remarkably different adhesion propensities. Moreover, introduction of a bifurcation as opposed to the commonly used linear channel resulted in significantly different flow and adhesion behaviors, which may have important implications in correlating these results to in vivo behavior. This study provides valuable information for design of carriers for targeted drug delivery.

Macrophages recognize size and shape of their targets.

Recognition by macrophages is a key process in generating immune response against invading pathogens. Previous studies have focused on recognition of pathogens through surface receptors present on the macrophage's surface. Here, using polymeric particles of different geometries that represent the size and shape range of a variety of bacteria, the importance of target geometry in recognition was investigated. The studies reported here reveal that attachment of particles of different geometries to macrophages exhibits a strong dependence on size and shape. For all sizes and shapes studied, particles possessing the longest dimension in the range of 2-3 microm exhibited highest attachment. This also happens to be the size range of most commonly found bacteria in nature. The surface features of macrophages, in particular the membrane ruffles, might play an important role in this geometry-based target recognition by macrophages. These findings have significant implications in understanding the pathogenicity of bacteria and in designing drug delivery carriers.

Endocytosis and Intracellular Distribution of PLGA Particles in Endothelial Cells: Effect of Particle Geometry.

Targeting, internalization, and intracellular trafficking of carriers are key processes in drug delivery to endothelial cells. We synthesized PLGA particles with spherical and elliptical disk geometries and investigated the effect of particle shape on rate of particle endocytosis and their intracellular distribution in endothelial cells. Elliptical disks (aspect ratio of 5) were endocytosed at a slower rate compared to spheres (1.8 µm diameter) of the same volume. However, both particles were eventually internalized and accumulated around the nucleus. We quantified the orientation of elliptical disks and found that disks, on average, oriented tangentially with the nuclear membrane. The non-spherical geometry of elliptical disks brings unique aspects to the kinetics and equilibrium distribution of these particles in cells.

Red blood cell-mimicking synthetic biomaterial particles.

Biomaterials form the basis of current and future biomedical technologies. They are routinely used to design therapeutic carriers, such as nanoparticles, for applications in drug delivery. Current strategies for synthesizing drug delivery carriers are based either on discovery of materials or development of fabrication methods. While synthetic carriers have brought upon numerous advances in drug delivery, they fail to match the sophistication exhibited by innate biological entities. In particular, red blood cells (RBCs), the most ubiquitous cell type in the human blood, constitute highly specialized entities with unique shape, size, mechanical flexibility, and material composition, all of which are optimized for extraordinary biological performance. Inspired by this natural example, we synthesized particles that mimic the key structural and functional features of RBCs. Similar to their natural counterparts, RBC-mimicking particles described here possess the ability to carry oxygen and flow through capillaries smaller than their own diameter. Further, they can also encapsulate drugs and imaging agents. These particles provide a paradigm for the design of drug delivery and imaging carriers, because they combine the functionality of natural RBCs with the broad applicability and versatility of synthetic drug delivery particles.