Cholestyramine, the first bile acid sequestrant to be marketed, has been in use for over 20 years. Despite its low potency, requiring 16-24 g of polymer to achieve 20% LDL cholesterol reduction in hypercholesterolemic individuals, only one other sequestrant, colestipol, has come to market in the ensuing period. GelTex Pharmaceuticals has been involved for over six years in the discovery and development of new, more potent polymeric sequestrants. Two binding mechanisms are presented — one that operates via an aggregate binding structure and one that is effective via a defined site binding structure. These two binding mechanisms are compared and contrasted through bile acid binding isotherms. The best of these new sequestrants bind bile acids through a combination of hydrophobicity and ion exchange. Optimization and balancing of each of these interactions led us to more potent materials. The first of these, colesevelam hydrochloride is expected to be three to four times more potent than cholestyramine. A third generation product is still in research at GelTex. With another twofold increase in potency possible, single tablet therapy may become a reality.

Despite tremendous progress towards a better understanding of polymer erosion, degradable polymers could so far not be classified according to their erosion behavior. It was the goal of this study to close this gap and to find those parameters that determine how a polymer erodes. Therefore, a dimensionless erosionnumber ε was defined, that can be calculated from the velocity of polymer degradation and water diffusion in the polymer bulk. s indicates whether erosion is a surface phenomenon (surface erosion) or whether it affects the complete polymer bulk bulk erosion). ε is characteristic for a polymer type and depends on the diffusivity of water inside polymer matrix, on the molecular weight of a polymer, and the dimensions of an eroding polymer matrix. The proposed erosion theory shows that ‘surface erosion’ and ‘bulk erosion’ are two potential erosion pathways that are open to any degradable polymer.

Recombinant DNA methods were used to create a new class of artificial proteins that undergo reversible gelation in response to changes in pH or temperature. These proteins consist of terminal a-helical “leucine zipper” domains flanking a central, water-soluble polvelectrolyte segment. The formation of coiled-coil aggregates of the terminal domains in near-neutral pH solution triggers formation of a polymer hydrogel, with the central polyelectrolyte segment retaining solvent and preventing precipitation of the chains. Dissociation of the coiled-coil aggregates through elevation of pH or temperature causes dissolution of the gel and a return to the viscous behavior characteristic of a polymer solution. The pH and temperature range of the hydrogel state and its viscoelastic properties may be systematically varied through precise changes of the length, composition and charge density of the terminal and central blocks. Such control is of value in designing hydrogels with predetermined physical properties and makes these biosynthetic triblock copolymer systems attractive candidates for use in molecular and cellular encapsulation and in controlled reagent delivery.

The grafting of poly (acrylic acid) and /or acrylamide onto biopolymer chitosan were carried in order to obtain hydrogel materials- The grafting reactions were carried out in a homogeneous aqueous phase by using potassium persulfate in combination with ferrous ammonium sulfate as redox initiator system in the case of acrylic acid and potassium persulfate alone in the case of acrylamide The percentage of grafting was found to depend on the relative amount of monomer to chitosan, initiator and ferrous ion concentration, volume of the aqueous phase as well as reaction time and reaction temperature. In the case of acrylic acid a maximum grafting of 1800% was obtained, whereas with acrylamide the optimum percentage of grafting attained was near 400%. Evidence of grafting was obtained from comparison of FTIR of the grafted and ungrafted chitosan as well as solubility characteristics and swelling behavior of the products. The swelling degree of the grafted chitosan samples was determined at two different pH values. The percentage of grafting influenced the degree of swelling of the grafted chitosan, in different manner depending on the nature of the grafted chains, where values as high as 40 was observed.

The dissolution of a drug delivery system consisting of gentamicin sulfate in a hydrophobic polyanhydride matrix has been examined. The in vitro release of gentamicin is a function of the composition of the dissolution medium, with slower release in pH 7.4 buffer than in unbuffered water. This is consistent with an anion exchange taking place under conditions in which carboxylate polymer chain-ends form a poorly soluble salt with gentamicin, and sulfate is released into solution. Results of additional experiments probing this model are digeussed.

Dark ground optical microscopy, electron microscopy, and protein release rate studies have been used to quantify the effects of formulation changes on the phase inversion dynamics and in vitro drug release properties of an injectable PLGA-based drug delivery system. Gel growth rates and water influx rates are determined from plots of the square of the respective front with time. Results show that additives that increase the solution gelation rate and produce finger-like void morphologies result in higher initial release rates. Conversely, additives that slow the rate of gelation dramatically reduce the initial drug release rate and lead to a more dense sponge-like morphology.

Major challenges in oral delivery of peptides include the need to overcome gastric and intestinal degradation. pH -Sensitive hydrogels are suitable candidates for oral drug delivery of peptides due to their ability to respond to their environment. We have developed a new type of hydrogel composed of poly(methacrylic acid) grafted with poly(ethylene glycol) which can be used as drug delivery carriers for salmon calcitonin. These hydrogels were prepared by free radical solution polymerization and were molecularly designed to contain poly(ethylene glycol) tethered chains promoting mucosal adhesion and providing calcitonin protection, as well as methacrylic acid moieties, which act as calcium binders leading to epithelial cell junction opening. Solutions of approximately 0.1 mg/ml of salmon calcitonin were used to load the protein into the gels at pH = 7 and constant ionic strength of 0.1 M. In vitro release studies were performed at pH=7 and 37 °C, while keeping an ionic strength of 0.1 M. Calcitonin release was achieved. The release behavior was explained in terms of diffusional theories.

Bioadhesive polymers are useful as drug delivery systems designed to adhere to the gastrointestinal lumen. The interaction between polymer and mucosal tissue influences residence time of the polymeric device and greatly affects the bioavailability of encapsulated drug. Poly(fumaric-co-sebacic anhydride) [P(FA:SA)] demonstrated impressive results in a variety of in vitro and in vivo experiments designed to test bioadhesion of microspheres. Among different molar ratios of P(FA:SA), adhesive measurements increased with increasing fumaric acid (FA) content. Using a modified microbalance technique, P(FA:SA)10:90 yielded a tensile work measurement of 82.99±12.76 nJ (mean±SEM) while that for P(FA:SA)70:30 was 453.23±47.73 nJ. A low molecular weight substance, fumaric anhydride prepolymer (FAPP), the oligomer form of fumaric acid, was incorporated into the microspheres and greatly increased the bioadhesive properties of P(FA:SA) as well as those of the relatively non-bioadhesive polymer poly(caprolactone) (PCL). Tensile work of P(FA:SA)20:80 was 32.95±5.42 nJ, and P(FA:SA)20:80 with 25% FAPP yielded a tensile work measurement of 556.28±113.12 nJ. Adhesion testing with PCL yielded a tensile work measurement of 7.93±1.84 nJ, while that for PCL with 25% FAPP was 1629.54±307.55 nJ. The effect of FA on drug delivery was evaluated in vitro using the P(FA:SA):FAPP blend with the everted intestinal sac technique. A low molecular weight drug (sodium salicylate) encapsulated in P(FA:SA)20:80 passed through the everted intestinal lumen to the interior of the sac to yield a concentration of 11.17±0.93 mg/dL, and drug encapsulated in P(FA:SA)20:80 with 10% FAPP yielded a concentration of 16.25±1.68 mg/dL (P<0.05). Unencapsulated drug passed through the intestinal lumen to yield a concentration of 8.09±-0.36 mg/dL. These experiments demonstrate that fumaric anhydride could be a very important component in bioadhesive polymer systems.

Novel barium sulphate preparations for X-ray contrasting with defined rheological properties have been developed which provide both low viscosity and high density. The relationship between volume fraction and both high-shear and low-shear viscosity was investigated to obtain the relative viscosity [η] and the limiting packing fraction Φm. The particles in a good preparation have a hydrophilic surface which allows the particles to penetrate through the mucus layer of the gastro-intestinal tract. Adsorption isotherms for chondroitin sulphate on barium sulphate from aqueous solution have been determined by spectrophotometry. FTIR spectroscopy and SEM has been used to analyse adsorption behaviour at a molecular level. Coating studies on mouse intestinal mucosa were carried out. A model for testing the coating thickness and particle packing of proprietary and test suspensions was developed. Surface and cross-sections characterisation of a novel test preparation on the intestinal model were determined using cryo scanning electron microscopy. In order to test the resolution obtained with novel material a phantom has been developed.

Particle uptake into intestinal tissue has seen increasing attention due to its implications in drug delivery. We attempted to observe a delivery system in vivo and examine uptake in different species. Microspheres were fabricated from polymers including polyanhydrides and delivered to an isolated loop of intestine in several species. The microspheres contained a dye either conjugated to a protein or incorporated freely and were used to qualitatively detect and locate the spheres in the villi of the length of the small intestine. Microspheres were dispersed, sized by a Coulter particle size analyzer, and characterized by confocal and cross-polarized light microscopy, FTIR and SEM. Coulter analysis revealed microspheres to be generally less than 5 microns in diameter. SEM typically showed homogeneous morphology among groups of microspheres. In vivo uptake experiments were performed in rodents, pigs, and ruminants using various microsphere formulations. Microspheres were delivered into the proximal end of the jejunum of anesthetized animals and allowed adequate transit time to be taken up. Animals were euthanized at various time points for explantation of tissue and sampling of blood. Excised samples were embedded inq polyvinyl alcohol, frozen, and cut into sections ranging between 7 and 14 μm in thickness. Our method of incorporating dyes allowed for simultaneous visualization by visible light microscopy and confocal laser scanning microscopy. Two-fluorochrome fluorescence of the microspheres and optical sectioning confirmed the presence of microspheres within intestinal tissue. The amount of uptake depended on the animal model, the duration of the experiment, and the composition of the microsphere. An assay for either the fluorescent dye, the protein attached to it, or the polymer encapsulating it may enable us to determine intracellular concentrations of mierospheres for the quantification of uptake.

Cytokines are ideal candidates for controlled release systems; they typically have short half-lives in vivo and may be more effective with localized delivery. A novel microencapsulation technique, phase inversion nanoencapsulation (PIN), was used to encapsulate interleukin-2 or interleukin-12 in biodegradable polymer microspheres. Cytokine was quantified by ELISA and bioactivity analyzed using a murine cytotoxic Tcell line proliferation assay. The release rates of IL-2 was dependant on release buffer components; inclusion of 10% fetal calf serum resulted in significantly higher release rates compared to plain buffer. Encapsulated IL-2 remained stable at all timepoints assayed (up to 4 months) when stored at 4 °C. Results indicated that PIN can be used to encapsulate IL-2 and IL-12, which were released throughout the duration of the release study (up to one month). These microsphere formulations have shown tumor suppressive affects in vivo and may provide a viable alternative to gene transfer for cancer immunotherapy.

Membrane-reservoir controlled release systems have been used for the delivery of a wide variety of therapeutic agents. Drug permeation is typically achieved by a solution-diffusion type of mechanism from a loaded reservoir region through a liquid-filled pore, and the rate of release is generally dependent on membrane porosity, tortuosity, and surface area. We have exploited the partition coefficient (between the reservoir liquid and the pore liquid phases) as an additional rate-controlling mechanism, and we have also extended the applications of membrane-reservoir systems to include delivery of microstructures such as liposomes.

If the fluid that fills the pore of the membrane is chosen to be immiscible with the liquid in the reservoir phase, the drug partitions between the two phases at the interface between the reservoir and the pore. The partition coefficient may be exploited to provide rate control by reducing the driving force for diffusion across the fluid-filled membrane pore. The performance of this type of system is evaluated using benzoic acid in an organic reservoir phase, diffusing through water-filled membrane pores. Both hydrophilic and hydrophobic membrane materials were investigated. A hydrophilic membrane material afforded the advantage of a more stable water-filled pore phase.

Controlled delivery of liposomal structures may also be achieved using a porous membranereservoir device. The pores of the membrane must be large enough to permit the passage of the liposomes, which are transported by simple Brownian motion. This concept is demonstrated using a porous polycarbonate membrane system, which was chosen to minimize interaction between the membrane and the liposomes.

We have used Small-Angle Neutron Scattering to investigate the structure of bilayer lipid membranes in aqueous solution. The lipids consist of equimolar concentrations of two molecules DMPG and DMPC (see text). The structures consist of solvent filled cores surrounded by shells composed of the lipid bilayers. In particular the radii of the core and shell thickness are measured as a function of lipid concentration and temperature. Other features which reveal themselves are vesicle forming ability of the lipids, additional larger structures and inter-vesicle interactions at large vesicle concentrations. The study is motivated by the possible use of these systems as delivery vehicles for various beneficial therapeutic compounds.

The interactions between dipalmitoylphosphocholine (DPPC) vesicles and a hyperbranched polymer micelle, Core(hex)PEG5, were examined by visual observation, differential scanning calorimetry (DSC), fluorescence microscopy and dynamic light scattering (DLS). The results from the DSC experiments showed that introducing the polymer to DPPC small unilamellar vesicles completely transformed the vesicles into a large unilamellar structure within minutes, a process that normally takes 2 days at room temperature. The hyperbranched polymers transformed the liposomes to the thermodynamically stable state. Fluorescence microscopy experiments showed that the DPPC vesicles became larger after the polymers were added. In addition, the polymers prevented aggregation of the liposome vesicles yielding clear solutions for up to two weeks at room temperature. In contrast, DPPC vesicles without polymers aggregated to form precipitate within hours at room temperature. The average sizes of liposomes and liposome/polymer were determined by DLS measurement with diameters of 36.3 nm and 65.2 nm, respectively.

Inhalation of dry powders comprised of large porous particles appears to promote long therapeutic action and increased systemic bioavailability (1-3). Relative to standard inhalation aerosols, dry powders possessing large porous particles clear relatively slowly from the lungs once inhaled (1) and show less aggregation prior to delivery. The diminished aggregation properties of large porous particles permits their use in a simple (and inexpensive) device without the need for carrier particles.

The feasibility of producing relatively monodisperse and biologically active protein particles by electrospray drying is demonstrated. The process entails dissolving dry powder in an electrosprayable solution. The solution is then dispersed and, after solvent evaporation, dry residues can be collected on suitable deposition substrates. The process was demonstrated in the case of insulin. Particles were sized visually, using a scanning electron microscope (SEM), and aerodynamically, using an inertial impactor. When electrosprays of nearly saturated solutions were operated in the stable cone-jet mode, impactor data showed that the particle average aerodynamic diameter ranged from about 88 to 110 nm in diameter and the distributions were quasi-monodisperse with relative standard deviation estimated at approximately 10%. SEM observations for the same conditions showed average particle dimensions ranging from 98 to 117 nm, with predominantly doughnut shapes. Smaller particles can be generated by decreasing the insulin concentration and/or by spraying smaller liquid flow rates. The biological activity of the electrospray-processed insulin samples was confirmed by comparing binding properties on insulin receptors against a control sample. Although the maximum production rate for monodisperse insulin nanoparticles from a single cone-jet is low, at about 0.23 mg/hour, overall production can be increased by multiplexing the device with microfabrication techniques.

Our spinning disk atomization (SDA) can, relative to other existing techniques, produce micronsized particles of very narrow size distribution. The aim of this work is to present this technology for the production of alginate microspheres. We have atomized and gelled aqueous alginate solutions into very narrowly dispersed microspheres with sizes ranging from 300 to 600 μm. Here the interest is to design particles of a given size with a narrow size distribution and to show a new method of encapsulation using the SDA. The viscosity and flow rate contributions in the drop formation is qualitatively analyzed to show how it affects the droplet size. In addition, a technique for high degree of encapsulation will be presented. As an example, yeast has been used as a model system. The production of yeast loaded microspheres will show the potential of the technique for biotechnology applications. Such alginate beads could potentially serve as carriers for acitive substances or cells in large scale bioreactors.

A method for preparation of hollow, biodegradable polymeric microcapsules for use as contrast agents is described, and strategies for their use in concomitant imaging and drug delivery are outlined. Compared with X-ray or magnetic resonance imaging, diagnostic ultrasound is a safe, relatively inexpensive imaging technique, which allows the physician to view real-time images. Contrast agents are being developed which will greatly enhance the contrast of the received image, when injected into the patients blood stream. We have developed hollow polymeric CA based upon the ability to microencapsulate a solid core of ammonium carbonate which is then removed by decomposition and freeze-drying. The polymer poly D,L(lactide-co-glycolide) (PLGA) was chosen for it's FDA approval, and because it has the most rapid in vivo degradation of the α-hydroxy acid series. Spray dried and solvent extraction samples were used to test the concept of concomitant drug delivery and imaging. The method of fabrication had an important effect on the drug loading by adsorption, and on the amount of drug that was released when the capsules were insonated with ultrasound in the medical imaging range. Both were greater for spray dried samples. Both frequency and pressure of insonation also influenced release. The most dramatic increase in release was after 5 minutes for the 10 Mhz low (1.25 MPa) pressure insonation, which showed 316% greater release than control, a total of 1.89 mg.

Bioactive materials such as calcium phosphate ceramics and bioactive glasses enhance bone tissue formation and then bond to bone tissue. In our work, we question what particular surface feature or features of bioactive materials are responsible for the bone tissue response. In this study we have uncoupled surface charge from surface chemistry, energy, and topography and have examined osteoblast adhesion to titanium surfaces of varying surface charge. We have shown that a negative surface charge promotes osteoblast adhesion by approximately 60% over a neutral surface and that conversely, a positive surface charge inhibits osteoblast adhesion by about 20%. Continued examination of surface characteristics that control cellular responses are warranted with the eventual goal of applying those desirable surface characteristics to any structural biomaterial for bone implant or tissue engineering applications.

The architecture of an engineered scaffold is an important consideration in the design of a synthetic tissue replacement. We have begun to explore the use of heirarchical fiber scale design ranging from microscale nonwoven to 3-D integrated fiber bundles in orthopaedic tissue engineering applications. The use of heirarchical fiber and design includes the following advantages: 1) the ability to tailor a broad spectrum of substrates with a wide range of mechanical properties, and 2) the ability to create structures capable of supporting cell proliferation as well as the micro-diffusion of nutrients throughout the structure. Using the bioresorbable copolymer, poly(D,L-lactide-co-glycolide) [PLAGA], we have fabricated two matrices based on fiber technology: 1) a microfiber non-woven mesh composed of 50:50 PLAGA and 2) a 3-dimensional braided structure composed of 5:95 PLAGA. These structures were examined by scanning electron microscopy [SEM] in an in vitro cell culture environment. Matrices were seeded with two types of cells (osteoblasts from neonatal rat calvaria and fibroblasts from the BALB/C cell line) to determine the effect of matrix architecture on cellular morphology and proliferation. SEM analysis of the microfiber matrix indicated a highly porous, structure resulting from the random arrangement of the microfibers. PLAGA fibers appear to have a range in diameter from approximately 2-7 μm. The 3-D braided matrix was shown to have a organized fibrous structure resulting from the 3-D braiding process. Examination of the attachment and proliferation of cells on the matrices revealed that cell morphology and proliferation patterns were dependent on cell type and matrix geometry. SEM analysis also indicated that cells responded dynamically to changes in structure due to the onset of degradation.