Biomedical subjects

Lonnie D Shea

Publications and source records attributed to Lonnie D Shea.

32 records · Page 2Linked to original sources

PubMed2005

Gene delivery through cell culture substrate adsorbed DNA complexes.

Efficient gene delivery is a fundamental goal of biotechnology and has numerous applications in both basic and applied science. Substrate-mediated delivery and reverse transfection enhance gene transfer by increasing the concentration of DNA in the cellular microenvironment through immobilizing a plasmid to a cell culture substrate prior to cell seeding. In this report, we examine gene delivery of plasmids that were complexed with cationic polymers (polyplexes) or lipids (lipoplexes) and subsequently immobilized to cell culture or biomaterial substrates by adsorption. Polyplexes and lipoplexes were adsorbed to either tissue culture polystyrene or serum-adsorbed tissue culture polystyrene. The quantity of DNA immobilized increased with time of exposure, and the deposition rate and final amount deposited depended upon the properties of the substrate and complex. For polyplexes, serum modification enhanced reporter gene expression up to 1500-fold relative to unmodified substrates and yielded equivalent or greater expression compared to bolus delivery. For lipoplexes, serum modification significantly increased the number of transfected cells relative to unmodified substrates yet provided similar levels of expression. Immobilized complexes transfect primary cells with improved cellular viability relative to bolus delivery. Finally, this substrate-mediated delivery approach was extended to a widely used biomaterial, poly(lactide-co-glycolide). Immobilization of DNA complexes to tissue culture polystyrene substrates can be a useful tool for enhancing gene delivery for in vitro studies. Additionally, adapting this system to biomaterials may facilitate application to fields such as tissue engineering.

Adsorption↗

PubMed2005

Crosslinked hyaluronic acid hydrogels: a strategy to functionalize and pattern.

The physiological activity of hyaluronic acid (HA) polymers and oligomers makes it a promising material for a variety of applications. The development of HA-hydrogel scaffolds with improved mechanical stability against degradation and biochemical functionality may enhance their application to tissue engineering. In this report, a crosslinking strategy targeting the alcohol groups via a poly(ethylene glycol) diepoxide crosslinker was investigated for the generation of degradable HA hydrogels. To provide support for cell adhesion in vitro, collagen was incorporated into the HA solution prior to the crosslinking process. The hydrogels have a continuous exterior and a porous interior, with pore diameters ranging from 6 to 9 microm. HA and HA-collagen hydrogels degrade in the presence of hyaluronidase and collagenase enzymes, indicating that the chemical modification does not prevent biodegradation. Complete degradation of the hydrogels occurred within 14 days in hyaluronidase (100 U/ml) and 3 days in collagenase (66 U/ml). Pattern transfer was employed to introduce a surface topography onto the hydrogel, which was able to orient cell growth. Furthermore, the hydrogels could be functionalized with the biomolecule neutravidin by incorporation of biotin along the HA backbone. This biotinylation approach may allow attachment of bioactive molecules that are conjugated to avidin.

Absorbable Implants↗

PubMed2005

DNA delivery from hyaluronic acid-collagen hydrogels via a substrate-mediated approach.

Efficient and controlled gene delivery from biodegradable materials can be employed to stimulate cellular processes that lead to tissue regeneration. In this report, a substrate-mediated approach was developed to deliver DNA from hyaluronic acid-collagen hydrogels. The hydrogels were formed by crosslinking HA with poly(ethylene glycol) diglycidyl ether. Poly(ethylene imine)(PEI)/DNA complexes were immobilized to the substrate using either biotin/neutravidin or non-specific adsorption. Complexes were formed in the presence or absence of salt to regulate complex size, and resulted in complexes with z-average diameters of 1221.7 +/- 152.3 and 139.4 +/- 1.3 nm, respectively. During 48-h incubation in PBS or hyaluronidase, DNA was released slowly from the hydrogel substrate (<30% of immobilized DNA), which was enhanced by incubation with conditioned media (approximately 50% of immobilized DNA). Transgene expression mediated by immobilized, large diameter complexes was 3 to 7-fold greater than for small diameter complexes. However, the percentage of cells expressing the transgene was greater for small diameter complexes (48.7%) than for large diameter complexes (22.3%). Spatially controlled gene transfer was achieved by topographically patterning the hydrogel to pattern cell adhesion. Biomaterial-based gene delivery can be applicable to numerous tissue engineering applications, or as a tool to examine tissue formation.

Absorbable Implants↗

PubMed2005

Plasmid delivery in vivo from porous tissue-engineering scaffolds: transgene expression and cellular transfection.

Tissue engineering scaffolds capable of sustained plasmid release can promote gene transfer locally and stimulate new tissue formation. We have investigated the scaffold design parameters that influence the extent and duration of transgene expression and have characterized the distribution of transfected cells. Porous scaffolds with encapsulated plasmid were fabricated from poly(lactide-co-glycolide) with a gas foaming procedure, with wet granulation employed to mix the components homogeneously prior to foaming. Wet granulation enhanced plasmid incorporation relative to standard procedures and also enhanced in vivo transgene expression, possibly through the increased loading and maintenance of the scaffold pore structure. The plasmid loading regulated the quantity and duration of transgene expression, with expression for 105 days achieved at the highest dosage. Expression was localized to the implantation site, though the distribution of transfected cells varied with time. Transfected cells were initially observed at the scaffold periphery (day 3), then within the pores and adjacent to the polymer (day 17), and finally throughout the scaffold interior (day 126). Delivery of a plasmid encoding VEGF increased the blood vessel density relative to control. Correlating scaffold design with gene transfer efficiency and tissue formation will facilitate application of plasmid-releasing scaffolds to multiple tissues.

Animals↗

PubMed2004

Delivery systems for small molecule drugs, proteins, and DNA: the neuroscience/biomaterial interface.

Manipulation of cellular processes in vivo by the delivery of drugs, proteins or DNA is of paramount importance to neuroscience research. Methods for the presentation of these molecules vary widely, including direct injection (either systemic or stereotactic), osmotic pump-mediated chronic delivery, or even implantation of cells engineered to indefinitely secrete a factor of interest. Biomaterial-based delivery systems represent an alternative to more traditional approaches, with the possibility of increased efficacy. Drug-releasing biomaterials, either as injectable microspheres or as three-dimensional implants, can deliver a molecule of interest (including small molecule drugs, biologically active proteins, or DNA) over a more prolonged period of time than by standard bolus injection, avoiding the need for repeated administration. Furthermore, sustained-release systems can maintain therapeutic concentrations at a target site, thus reducing the chance for toxicity. This review summarizes applications of polymer-based delivery of small molecule drugs, proteins, and DNA specifically relevant to neuroscience research. We detail the fabrication procedures for the polymeric systems and their utility in various experimental models. The biomaterial field offers unique experimental tools with downstream clinical application for the study and treatment of neurologic disease.

Animals↗

PubMed2004

Modular design of non-viral vectors with bioactive components.

Inefficient gene delivery continues to limit gene therapy applications to both basic and applied sciences. Approaches for engineering vectors increasingly include bioactive components that bind cellular receptors, disrupt membranes, or enhance nuclear transport. Recently, a novel cationic lipid was developed by modifying the glucocorticoid dexamethasone. This cationic corticosteroid condenses plasmids for gene delivery, while also modulating inflammation. Modular vectors containing bioactive components that target various cellular processes can overcome the barriers limiting gene transfer.

Active Transport, Cell Nucleus↗

PubMed2004

Controlled release systems for DNA delivery.

Adapting controlled release technologies to the delivery of DNA has the potential to overcome extracellular barriers that limit gene therapy. Controlled release systems can enhance gene delivery and increase the extent and duration of transgene expression relative to more traditional delivery methods (e.g., injection). These systems typically deliver vectors locally, which can avoid distribution to distant tissues, decrease toxicity to nontarget cells, and reduce the immune response to the vector. Delivery vehicles for controlled release are fabricated from natural and synthetic polymers, which function either by releasing the vector into the local tissue environment or by maintaining the vector at the polymer surface. Vector release or binding is regulated by the effective affinity of the vector for the polymer, which depends upon the strength of molecular interactions. These interactions occur through nonspecific binding based on vector and polymer composition or through the incorporation of complementary binding sites (e.g., biotin-avidin). This review examines the delivery of nonviral and viral vectors from natural and synthetic polymers and presents opportunities for continuing developments to increase their applicability.

DNA↗

PubMed2004

Gene delivery from polymer scaffolds for tissue engineering.

The combination of gene therapy with tissue engineering offers the potential to direct progenitor cell proliferation and differentiation into functional tissue replacements. Many approaches to engineering tissue replacements feature a polymer scaffold to create and maintain a space, support cell adhesion, and organize tissue formation. Polymer scaffolds, either natural, synthetic, or a combination of the two, have also been adapted to serve as delivery vehicles for viral and nonviral vectors, which can induce the expression of tissue inductive factors. Gene delivery is a versatile approach, capable of targeting any cellular process through localized expression of tissue inductive factors. The design and application of tissue engineering scaffolds for localized gene transfer are reviewed. Scaffolds are designed either to release the vector into the local tissue environment or maintain the vector at the polymer surface, which is regulated by the effective affinity of the vector for the polymer. Polymeric delivery can enhance gene transfer locally, promote and extend transgene expression, avoid vector distribution to distant tissues, and reduce the immune response to the vector. Scaffolds capable of controlled DNA delivery can provide a fundamental tool for directing progenitor cell function, which has applications with the engineering of numerous types of tissue. The utility of this approach will increase with the development of design parameters that correlate release and transgene expression, and with continued research into the biology of tissue formation.

Cell Culture Techniques↗

PubMed2003

Substrate-mediated DNA delivery: role of the cationic polymer structure and extent of modification.

DNA complex immobilization to substrates that support cell adhesion can enhance gene transfer by maintaining DNA within the cellular environment while limiting complex aggregation. This report examines the tether design (e.g., extent of functionalization) and cationic polymer structure for their effect on complex binding to the substrate and cellular transfection. DNA is complexed with cationic polymers (polylysine, PL; polyethylenimine, PEI), which are functionalized with biotin for binding to a neutravidin (NA) substrate. Surfaces densities ranging from 0.4 to 2.6 microg DNA/cm(2) were obtained for PL, and from 0.7 to 1.0 microg DNA/cm(2) for PEI. The distribution of biotin groups for PL/DNA complexes had a dual effect on cellular transfection. Increasing the fraction of PL with biotin residues decreased luciferase activity; however, increasing the number of biotin residues per PL increased luciferase activity. For PEI, the number of biotin groups present on the complex did not affect transgene expression. Release studies demonstrated that 20-30% of the immobilized DNA was released over 8 days, with 8-20% released during the first 24 h. Enzymatic degradation of cationic polymers is not necessary for transfection. Additionally, the duration of transgene expression was extended for surface-mediated delivery relative to bolus delivery.

Cations↗

PubMed2003

Murine granulosa cell morphology and function are regulated by a synthetic Arg-Gly-Asp matrix.

Extracellular matrix (ECM) proteins are established regulators of granulosa cell survival, morphology, and differentiation. In this study, the roles of ECM adhesion peptide density on murine granulosa cell adhesion, morphology, and steroid secretion were probed using synthetic matrices. The synthetic matrix was fabricated from the polysaccharide alginate, which does not inherently support cell adhesion but can be modified with controlled densities of adhesion peptides (10(-4) to 2 x 10(-1) ng/cm(2)). GRM02, a murine granulosa cell line, and primary murine granulosa cells were cultured on alginate matrices modified by coupling of synthetic peptide sequences containing the Arg-Gly-Asp motif common to ECM proteins. Cells cultured on these peptide-modified surfaces (0.02, 0.2 ng/cm(2)) attached and spread, with morphologies specific to the peptide identity and density. Additionally, progesterone and estradiol secretion was a function of peptide density, with up to threefold increases compared to controls. These results indicate that the density and identity of adhesion peptides regulate granulosa cell function. This system provides a mechanism to examine the granulosa cell-ECM interactions that occur during follicle maturation.

Alginates↗

PubMed2003

Controllable delivery of non-viral DNA from porous scaffolds.

The inductive approach to tissue engineering combines three-dimensional porous scaffolds with drug delivery to direct the action of progenitor cells into a functional tissue. We present an approach to fabricate scaffolds capable of controlled, sustained delivery by the assembly and subsequent fusion of drug-loaded microspheres using a gas foaming/particulate leaching process. DNA-loaded microspheres were fabricated from the copolymers of lactide and glycolide (PLG) using a cryogenic double emulsion process. Microspheres were fabricated in four populations with mean diameters ranging from 12.3 microm to 92.5 microm. Scaffolds fabricated by fusion of these microspheres had an interconnected open pore structure, maintained DNA integrity, and exhibited sustained release for 21 days. Control over the release was obtained through manipulating the properties of the polymer, microspheres, and the foaming process. Decreasing the microsphere diameter or the molecular weight of the polymer used for microsphere fabrication led to increased rates of release from the porous scaffold. Additionally, increasing the pressure of CO(2) increased the DNA release rate. The ability to create porous polymer scaffolds capable of controlled release rates may provide a means to enhance and regulate gene transfer within a developing tissue, which will increase their utility in tissue engineering.

DNA↗

PubMed2003

Novel approach for the three-dimensional culture of granulosa cell-oocyte complexes.

The in vitro culture of immature ovarian follicles is used to examine the factors that regulate follicle development and may ultimately provide options for reproductive infertility. The objective of this study was to develop a three-dimensional in vitro culture system for the growth and development of individual granulosa cell-oocyte complexes. An alginate hydrogel was used to encapsulate immature mouse granulosa cell-oocyte complexes (GOCs) that were subsequently maintained in a serum-free in vitro culture. An overall incorporation efficiency of 50% was achieved. The complexes were assessed by transmission electron microscopy for changes in ultrastructure during in vitro growth. The architecture of the follicular complex was maintained during the encapsulation and the subsequent culture. The granulosa cells proliferated, and the oocytes also grew in volume and obtained the structural characteristics of mature oocytes including cortical granule formation, a well-developed zona pellucida with microvilli, normal mitochondria, and lattice-like structures in the cytoplasm. Oocytes retrieved and matured were able to resume meiosis, a necessary step for proper development. Thus, this system represents a new in vitro methodology for growth of individual granulosa cell-oocyte complexes.

Alginates↗

PubMed2002

Drug-releasing scaffolds fabricated from drug-loaded microspheres.

Biodegradable scaffolds serve a central role in many strategies for engineering tissue replacements or in guiding tissue regeneration. Typically, these scaffolds function to create and maintain a space and to provide a support for cell adhesion. However, these scaffolds also can serve as vehicles for the delivery of bioactive factors (e.g., protein or DNA) in order to manipulate cellular processes within the scaffold microenvironment. This study presents a novel approach to fabricate tissue-engineering scaffolds capable of sustained drug delivery whereby drug-loaded microspheres are fabricated into structures with controlled porosity. A double-emulsion process was used to fabricate microspheres with encapsulated DNA that retained its integrity and was released from the microspheres within 24 h. These DNA-loaded microspheres subsequently were formed into a nonporous disk or an interconnected open-pore scaffold (>94% porosity) via a gas-foaming process. The disks and scaffolds exhibited sustained plasmid release for at least 21 days and had minimal burst during the initial phase of release. This approach of assembling drug-loaded microspheres into porous and nonporous structures may find great utility in the fabrication of synthetic matrices that direct tissue formation.

Biocompatible Materials↗

PubMedDate not supplied

Surface-tethered DNA complexes for enhanced gene delivery.

Overcoming the barriers to efficient gene transfer is a fundamental goal of biotechnology. A versatile approach to enhance the delivery of nonviral DNA involves complexation with cationic polymers, which can be designed to overcome the barriers to effective gene transfer. More recently, DNA release from a polymer substrate or scaffold has been shown to enhance gene transfer, likely by increasing DNA concentrations in the cell microenvironment. We propose a novel approach that combines these two strategies in which cationic polymer/DNA complexes are tethered to a substrate that supports cell adhesion. The cationic polymers package the DNA for efficient internalization and the surface tethering functions to maintain elevated concentrations in the cell microenvironment for cells adhered to the substrate. The cationic polymer polylysine (degree of polymerization equal to 19 or 150) was modified with biotin groups, which was confirmed by mass spectrometry and biochemical analysis. Complex formation of DNA with biotinylated-polylysine, or mixtures of biotinylated and nonbiotinylated polylysines, was confirmed by gel electrophoresis. Plasmid DNA encoding for the reporter gene beta-galactosidase was complexed with different mixtures of biotinylated and nonbiotinylated polylysine and incubated on neutravidin (nonglycosylated avidin)-coated surfaces. DNA surface densities ranging from 0.1 to 4.3 microg/cm2 were observed and found to be a function of the number of biotin groups, the molecular weight of the polylysine, and the amount of DNA. HEK293T or NIH/3T3 cells were then seeded onto the DNA-modified surfaces, and transfection was quantified at 48 and 96 h. Transfection by the DNA surfaces was observed with both cell lines, and expression levels up to 100 fold greater than bulk delivery of the complexes was obtained. Transfection was found to be a function of the surface DNA quantities and the number of tethers on the complex. Transfected cells were observed only in the region in which DNA complexes were tethered, suggesting that the location of transfected cells can be specifically controlled. Surface tethering of DNA represents a promising approach to enhancing gene transfer and spatially controlling gene delivery, which may have applications to a multitude of fields ranging from tissue engineering to functional genomics.

3T3 Cells↗