Electrospinning Drug-Loaded Alginate-Based Nanofibers towards Developing a Drug Release Rate Catalog.

Electrospinning natural polymers represents a developing interest in the field of biomaterials. Electrospun nanofibers have been shown to facilitate tissue regeneration and emulate body tissue, making them ideal for modern biomedical applications. These water-soluble natural polymers including alginate, have also shown promise as drug delivery vehicles. However, many biopolymers including alginate are inherently charged, making the formation of nanofibers difficult. To better understand the potential of natural polymer-based fibers in drug delivery applications, fiber formulations and drug loading concentrations of alginate-based scaffolds were investigated. It was found electrospinning poly(vinyl alcohol) with alginate facilitated fiber formation while the co-polymer agarose showed minor improvement in terms of alginate electrospinnability. Once uniform fibers were formed, the antibiotic ciprofloxacin was added into the polymer electrospinning solution to yield drug-loaded nanofibers. These optimized parameters coupled with small molecule release rate data from the drug-loaded, alginate-based fibers have been used to establish a catalog of small molecule release profiles. In the future, this catalog will be further expanded to include drug release rate data from other innately charged natural polymer-based fibers such as chitosan. It is anticipated that the cataloged profiles can be applied in the further development of biomaterials used in drug delivery.

Development of 3D hydrogel culture systems with on-demand cell separation.

Recently there has been an increased interest in the effects of paracrine signaling between groups of cells, particularly in the context of better understanding how stem cells contribute to tissue repair. Most current 3D co-culture methods lack the ability to effectively separate two cell populations after the culture period, which is important for simultaneously analyzing the reciprocal effects of each cell type on the other. Here, we detail the development of a 3D hydrogel co-culture system that allows us to culture different cell types for up to 7 days and subsequently separate and isolate the different cell populations using enzyme-sensitive glues. Separable 3D co-culture laminates were prepared by laminating PEG-based hydrogels with enzyme-degradable hydrogel adhesives. Encapsulated cell populations exhibited good segregation with well-defined interfaces. Furthermore, constructs can be separated on-demand upon addition of the appropriate enzyme, while cell viability remains high throughout the culture period, even after laminate separation. This platform offers great potential for a variety of basic cell signaling studies as the incorporation of an enzyme-sensitive adhesive interface allows the on-demand separation of individual cell populations for immediate analysis or further culture to examine persistence of co-culture effects and paracrine signaling on cell populations.

Development of nano- and microscale chondroitin sulfate particles for controlled growth factor delivery.

Size scale plays an important role in the release properties and cellular presentation of drug delivery vehicles. Because negatively charged chondroitin sulfate (CS) is capable of electrostatically sequestering positively charged growth factors, CS-derived nanoscale micelles and microscale spheroids were synthesized as potential growth factor carriers to enhance differentiation of stem cells. Particles were characterized for morphology, size distribution, surface charge and cytocompatibility, as well as release of transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α). CS micelles were spherical and negatively charged with a bimodal distribution of 324.1±8.5 and 73.2±4.4 nm diameters, and CS microspheres possessed a rounded morphology and a diameter of 4.3±0.93 µm. Positively charged TGF-ß1 demonstrated minimal release after loading in CS microspheres, while negatively charged TNF-α exhibited substantial release over the first 15 h, suggesting that TGF-ß1 electrostatically complexed with CS. The micelles and microparticles were found to be cytocompatible at moderate concentrations with marrow stromal cell monolayers and within embryonic stem cell embryoid bodies. These synthesis techniques, which allow the formation of CS-based carriers over a variety of nano- and microscale sizes, offer versatility for tailored release of positively charged growth factors and controlled CS presentation for a variety of stem cell-based applications in tissue engineering and regenerative medicine.

Molecular dendritic transporter nanoparticle vectors provide efficient intracellular delivery of peptides.

We present the synthesis of a modular delivery system that is composed of two main macromolecular building blocks, dendritic molecular transporter molecules and a polymeric scaffold in a size dimension of 5-10 nm. The conjugated dendritic molecular transporter units proved to be critical for the delivery of the polymer nanoparticle into 3T3 cells and illustrates the dendritic molecular transporter promoted intracellular uptake of polymer particles derived from intramolecular chain collapse processes. In a sequence of modification steps, pyridinyldithio linker was introduced to undergo thiol-disulfide exchange reactions with peptide sequences containing cysteine amino acid units to furnish peptide-nanoparticle conjugates with cleavable disulfide linkers. The intracellular uptake of the nanoparticle conjugates and the delivery of the peptidic cargo were studied via dual labeling of the nanoparticle with Alexa Fluor 568 dye and fluorescein (FITC) markers on the peptide in mammalian cell lines such as NIH 3T3 cells via confocal microscopy. In this work, we have demonstrated the assembly of a novel nanoscopic delivery system in which the conjugated dendritic molecular transporter molecules facilitated the rapid cellular uptake of a nanoparticle-peptide conjugate with up to 25 copies of peptidic cargo to establish new venues for the implementation of protein and oligonucleotide drugs.

Effective delivery of IgG-antibodies into infected cells via dendritic molecular transporter conjugate IgGMT.

IgG antibody-transporter conjugates enable intracellular uptake of biologically active IgG antibodies that inhibit viral mediated syncytia formation in respiratory syncytial virus green fluorescent protein (RSV-GFP) infected human epithelial cells (HEp-2).