Applications of lipid vesicles : drug delivery systems and templates for nanometer and micron sized structures.

The pH-dependent conformational transition of poly(2-ethylacrylic acid) [PEAA] was investigated by fluorescence spectroscopy using pyrene as a probe. We demonstrate that solvent fractionation is effective in reducing the polydispersity of a PEAA sample obtained from bulk free-radical polymerization, and in providing PEAA fractions of various molecular weights. The breadth of the conformational transition was reduced by using samples of lower polydispersity. Furthermore, the location of the conformational transition on the pH axis was shown to be dependent upon the molecular weight of the sample. The interaction of PEAA with phosphatidylcholine vesicles was studied. PEAA was shown to induce fusion of phosphatidylcholine bilayer membranes under mildly acidic conditions. The pH-dependent destabilization and fusion of extruded large unilamellar vesicles (LUVs) by PEAA was characterized by optical density measurements, transmission electron microscopy, and lipid mixing and contents release assays. Reduction of either the chain length or the polymer concentration caused the fusion and contents release events to shift to lower pH values. Release of entrapped calcein was observed at pH values ca. 1 unit higher than those found to cause membrane fusion. Decreased levels of fusion were observed when the concentration of PEAA was lower than that of the lipid; however, quantitative release of encapsulated calcein could be effected at very low polymer concentrations (∼3% w/w PEAA/lipid). Giant unilamellar vesicles were used as templates for producing flexible polymeric wires. Tubes up to several microns in diameter formed spontaneously upon dehydration of the vesicles, or were formed mechanically by shearing the vesicle to create the appropriate membrane instability. The resulting tubes were stabilized by photopolymerization of poly(ethylene glycol) dimethacrylate [PEGDMA], the lumenally confined macromonomer. A detailed study of the mechanical properties of crosslinked PEGDMA filaments was performed. Four different molecular weight PEGDMAs were synthesized from the corresponding dihydroxy terminated poly(ethylene glycol) and methacroyl chloride. Mechanical properties of bulk hydrogels were measured via tensile testing with an Instron, and these values are compared to those obtained on micron sized filaments connecting two spheres (hydrogel dumbbell) by stretching the dumbbells in a flow field. Furthermore, techniques were developed for drawing a nanotube (or an array of tubes) between gold contacts on a surface. We have also demonstrated the use of this templating approach to produce pH-responsive hydrogels composed of poly(ethylene glycol) dimethacrylate and methacrylic acid

Toward an Artificial Golgi: Redesigning the Biological Activities of Heparan Sulfate on a Digital Microfluidic Chip

Using digital microfluidics, recombinant enzyme technology, and magnetic nanoparticles, we have created a functional prototype of an artificial Golgi organelle. Analogous to the natural Golgi, which is responsible for the enzymatic modification of glycosaminoglycans immobilized on proteins, this artificial Golgi enzymatically modifies glycosaminoglycans, specifically heparin sulfate (HS) chains immobilized onto magnetic nanoparticles. Sulfo groups were transferred from adenosine 3′-phosphate 5′-phosphosulfate to the 3-hydroxyl group of the D-glucosamine residue in an immobilized HS chain using D-glucosaminyl 3-O-sulfotransferase. After modification, the nanoparticles with immobilized HS exhibited increased affinity for fluorescently labeled antithrombin III as detected by confocal microscopy. Since the biosynthesis of HS involves an array of specialized glycosyl transferases, epimerase, and sulfotransferases, this approach should mimic the synthesis of HS in vivo. Furthermore, our method demonstrates the feasibility of investigating the effects of multi-enzyme systems on the structure of final glycan products for HS-based glycomic studies

pH-Induced Fusion and Lysis of Phosphatidylcholine Vesicles by the Hydrophobic Polyelectrolyte Poly(2-ethylacrylic Acid)

Poly(2-ethylacrylic acid) [PEAA] was shown to induce fusion of phosphatidylcholine bilayer membranes under mildly acidic conditions. The pH-dependent destabilization and fusion of extruded large unilamellar vesicles (LUVs) by PEAA was characterized by optical density measurements, transmission electron microscopy, and lipid-mixing and contents-release assays. Reduction of either the chain length or the polymer concentration caused the fusion and contents-release events to shift to lower pH values. Release of entrapped calcein was observed at pH values approximately 1 unit higher than those found to cause membrane fusion. Decreased levels of fusion were observed when the concentration of PEAA was lower than that of the lipid; however, quantitative release of encapsulated calcein could be effected at very low polymer concentrations (∼3% w/w PEAA/lipid)

Free-Radical Synthesis of Poly(2-ethylacrylic acid) Fractions of Low Polydispersity: Effects of Molecular Weight and Polydispersity on the pH-Dependent Conformational Transition in Aqueous Solutions

The interaction of the hydrophobic polyelectrolyte, poly(2-ethylacrylic acid) [PEAA, 1], with phospholipid bilayer membranes has been studied extensively in this laboratory over the past fifteen years. This system has been tailored to create lipid vesicles that respond via release of contents to changes in pH, temperature, light intensity, or concentration of a solute such as glucose

Artificial Organelles: Digital Microfluidic Platform for Proteoglycan and Glycoprotein Biosynthesis

The development of artificial organelles is a new, fast-growing field in which a variety of techniques have been employed. This article gives a brief overview of the history of artificial organelles, and describes the development of an artificial endoplasmic reticulum and Golgi organelle on a digital microfluidic platform. This device should be useful in high-throughput combinatorial proteoglycan/glycoprotein synthesis, providing critical information about the control of biosynthetic pathways for these important signaling molecules

Bioengineering murine mastocytoma cells to produce anticoagulant heparin.

Heparin (HP), an important anticoagulant polysaccharide, is produced in a complex biosynthetic pathway in connective tissue-type mast cells. Both the structure and size of HP are critical factors determining the anticoagulation activity. A murine mastocytoma (MST) cell line was used as a model system to gain insight into this pathway. As reported, MST cells produce a highly sulfated HP-like polysaccharide that lacks anticoagulant activity (Montgomery RI, Lidholt K, Flay NW, Liang J, Vertel B, Lindahl U, Esko JD. 1992. Stable heparin-producing cell lines derived from the Furth murine mastocytoma. Proc Natl Acad Sci USA 89:11327-11331). Here, we show that transfection of MST cells with a retroviral vector containing heparan sulfate 3-O-sulfotransferase-1 (Hs3st1) restores anticoagulant activity. The MST lines express N-acetylglucosamine N-deacetylase/N-sulfotransferase-1, uronosyl 2-O-sulfotransferase and glucosaminyl 6-O-sulfotransferase-1, which are sufficient to make the highly sulfated HP. Overexpression of Hs3st1 in MST-10H cells resulted in a change in the composition of heparan sulfate (HS)/HP and CS/dermatan sulfate (DS) glycosaminoglycans. The cell-associated HS/HP closely resembles HP with 3-O-sulfo group-containing glucosamine residues and shows anticoagulant activity. This study contributes toward a better understanding of the HP biosynthetic pathway with the goal of providing tools to better control the biosynthesis of HP chains with different structures and activities