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Biomedical subjects

David M Lynn

Publications and source records attributed to David M Lynn.

At least 19 recordsLinked to original sources
PubMed2006

Heparin localization and fine structure regulate Burkitt's lymphoma growth.

Burkitt's lymphoma (BL) is a B-cell malignancy associated with the Epstein-Barr virus (EBV). Mounting evidence has implicated heparan sulfate proteoglycans and heparan sulfate-like glycosaminoglycans (HSGAGs) in the initiation, severity, and progression of the malignancy. The importance of HSGAGs in regulating BL cell growth was therefore examined. Extracellular exogenous heparin inhibited cell growth >30%, while heparin internalized with poly(beta-amino ester)s promoted proliferation up to 58%. The growth-modulating effects of heparin and internalized heparin were dependent on cell surface HSGAGs, PI3K, and Erk/Mek. Treatment of cells with protamine sulfate or with heparinases potently inhibited proliferation, with the greatest effects induced by heparinase I. Cell surface HSGAGs therefore play an important role in regulating BL proliferation and may offer a potential target for therapeutic intervention.

Burkitt Lymphoma↗
PubMed2006

Controlling interlayer diffusion to achieve sustained, multiagent delivery from layer-by-layer thin films.

We present the fabrication of conformal, hydrolytically degradable thin films capable of administering sustained, multiagent release profiles. Films are constructed one molecular layer at a time by using the layer-by-layer, directed-deposition technique; the subsequent hydrolytic surface erosion of these systems results in the release of incorporated materials in a sequence that reflects their relative positions in the film. The position of each species is determined by its ability to diffuse throughout the film architecture, and, as such, the major focus of this work is to define strategies that physically block interlayer diffusion during assembly to create multicomponent, stratified films. By using a series of radiolabeled polyelectrolytes as experimental probes, we show that covalently crosslinked barriers can effectively block interlayer diffusion, leading to compartmentalized structures, although even very large numbers of ionically crosslinked (degradable or nondegradable) barrier layers cannot block interlayer diffusion. By using these principles, we designed degradable films capable of extended release as well as both parallel and serial multiagent release. The ability to fabricate multicomponent thin films with nanoscale resolution may lead to a host of new materials and applications.

Journal Article↗
PubMed2006

Ferrocene-containing cationic lipids for the delivery of DNA: oxidation state determines transfection activity.

The ability of two redox-active, ferrocene-containing cationic lipids [11-(ferrocenylundecyl)trimethylammonium bromide (FTMA) and bis(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA)] to transfect mammalian cells was investigated. This study sought to determine the range of conditions over which these lipids were capable of transfecting cells and whether the oxidation state of the ferrocenyl groups in these materials could be used to influence the extent of transfection. Experiments conducted in the COS-7 cell line demonstrated that reduced and oxidized FTMA were substantially cytotoxic and did not transfect cells. Subsequent experiments conducted using BFDMA and reporter plasmids encoding enhanced green fluorescent protein (EGFP) and firefly luciferase demonstrated that BFDMA was able to transfect cells. However, the extent of transfection depended significantly upon both the concentration of BFDMA and the oxidation state of the lipid. Quantitative characterization of cytotoxicity and gene expression demonstrated that a window of concentration existed over which reduced BFDMA was non-cytotoxic and yielded high levels of transfection, but over which electrochemically oxidized BFDMA yielded very low (background) levels of transfection. Characterization of lipoplexes using dynamic light scattering demonstrated that reduced and oxidized BFDMA formed small aggregates (ca. 90 to 250nm) at concentrations of lipid ranging from 2 to 10 microM. Taken together, these results demonstrate that the oxidation state of BFDMA, which can be controlled electrochemically, can be used to control the extent of cell transfection. These results could form the basis of transfection procedures that exploit the redox behavior of ferrocene-containing lipids to achieve active spatial and temporal control over transfection using electrochemical methods.

Animals↗
PubMed2006

Structure/property relationships in erodible multilayered films: influence of polycation structure on erosion profiles and the release of anionic polyelectrolytes.

We have investigated the influence of polymer structure on the erosion profiles of multilayered polyelectrolyte assemblies fabricated from sodium poly(styrene sulfonate) (SPS) and three different hydrolytically degradable polyamines. We synthesized three structurally related poly(beta-amino ester)s (polymers 1-3) having systematic variations in both charge density and hydrophobicity. These changes in structure did not influence film thickness significantly, but polymer structure was found to play an important role in defining the rates at which multilayered assemblies fabricated from these materials eroded in physiologically relevant media. Films 60 nm thick fabricated from polymer 1 and SPS eroded completely in 50 h when incubated in PBS buffer at 37 degrees C, as determined by ellipsometry. Analogous films fabricated from polymers 2 and 3 eroded and released SPS into solution over significantly longer time periods ranging from approximately 150 h (ca. 6 days) to 370 h (ca. 15 days), respectively. These differences are consistent with a systematic increase in the hydrophobicity of polymers 1-3 as well as the relative rates at which these polymers degrade hydrolytically. This work demonstrates that it is possible to tailor the rates at which thin, multilayered polyelectrolyte assemblies release incorporated anionic polyelectrolytes over a large range of time periods simply by changing the structure of the degradable polyamine used to fabricate a film. The principles reported here may therefore contribute to the design of multilayered assemblies that permit a broad range of spatial and temporal control over the release of therapeutic agents from coated surfaces.

Coated Materials, Biocompatible↗
PubMed2006

Release of plasmid DNA from intravascular stents coated with ultrathin multilayered polyelectrolyte films.

Materials that permit control over the release of DNA from the surfaces of topologically complex implantable devices, such as intravascular stents, could contribute to the development of new approaches to the localized delivery of DNA. We report the fabrication of ultrathin, multilayered polyelectrolyte films that permit both the immobilization and controlled release of plasmid DNA from the surfaces of stainless steel intravascular stents. Our approach makes use of an aqueous-based, layer-by-layer method for the assembly of nanostructured thin films consisting of alternating layers of plasmid DNA and a hydrolytically degradable polyamine. Characterization of coated stents using scanning electron microscopy (SEM) demonstrated that stents were coated uniformly with an ultrathin film ca. 120 nm thick that adhered conformally to the surfaces of stent struts. These ultrathin films did not crack, peel, or delaminate substantially from the surface after exposure to a range of mechanical challenges representative of those encountered during stent deployment (e.g., balloon expansion). Stents coated with eight bilayers of degradable polyamine and a plasmid encoding enhanced green fluorescent protein (EGFP) sustained the release of DNA into solution for up to four days when incubated in phosphate buffered saline at 37 degrees C, and coated stents were capable of mediating the expression of EGFP in a mammalian cell line without the aid of additional transfection agents. The approach reported here could, with further development, contribute to the development of localized gene-based approaches to the treatment of cardiovascular diseases or related conditions.

Animals↗
PubMed2005

Formulation and characterization of poly (beta amino ester) microparticles for genetic vaccine delivery.

Microparticulate delivery systems are a promising and versatile enhancement to DNA vaccines because they can target large payloads of plasmid and immunomodulating materials to antigen presenting cells (APC). A pH sensitive poly-beta amino ester (PBAE) has been recently described which substantially increases adjuvancy and delivery efficiency of such microparticle formulations. This work describes the characterization and formulation considerations specific to these PBAE containing microparticles. PBAE increases the supercoiled content and overall effective loading of plasmid DNA. This polymer also significantly buffers the pH microenvironment created by ester bond degradation, rendering encapsulated plasmid more suitable for transfection. Release of plasmid from microparticles is controllable based on the amount of PBAE in the composition. Transfection is dependant upon phagocytosis, and is optimal for 15% and 25% PBAE microparticle formulations. However, larger quantities of PBAE may be toxic to cells indicating that the 15% PBAE formulation is a suitable candidate for delivery in future studies with disease specific, DNA vaccines.

Animals↗
PubMed2005

Ferrocene-containing cationic lipids: influence of redox state on cell transfection.

A ferrocene-containing, redox-active cationic lipid that can be transformed using electrochemical methods yields large differences in cell transfection depending on the oxidation state of the lipid. Expression of enhanced green fluorescent protein and firefly luciferase occurs at very high levels when DNA lipoplexes are formulated using the lipid in the reduced state. In contrast, transfection is negligible when oxidized lipid is used. These observations suggest the basis of a general method that could be used to transform inactive lipoplex formulations to an active form through the application of externally applied electrical potentials. The ability to activate lipoplexes toward transfection electrochemically and "on demand" could create new opportunities to deliver DNA in vitro and in vivo with both spatial and temporal control.

Animals↗
PubMed2005

Multilayered polyelectrolyte films promote the direct and localized delivery of DNA to cells.

Multilayered polyelectrolyte films fabricated from plasmid DNA and a hydrolytically degradable synthetic polycation can be used to direct the localized transfection of cells without the aid of a secondary transfection agent. Multilayered assemblies 100 nm thick consisting of alternating layers of synthetic polymer and plasmid DNA encoding for enhanced green fluorescent protein (EGFP) were deposited on quartz substrates using a layer-by-layer fabrication procedure. The placement of film-coated slides in contact with COS-7 cells growing in serum-containing culture medium resulted in gene expression in cells localized under the film-coated portion of the slides. The average percentage of cells expressing EGFP relative to the total number of cells ranged from 4.6% to 37.9%, with an average of 18.6%+/-8.2%, as determined by fluorescence microscopy. In addition to providing a mechanism for the immobilization of DNA at the cell/surface interface, a preliminary analysis of film topography by atomic force microscopy (AFM) demonstrated that polymer /DNA films undergo significant structural rearrangements upon incubation to present surface bound condensed plasmid DNA nanoparticles. These data suggest that the presence of the cationic polymer in these materials may also contribute to the internalization and expression of plasmid. The materials and design principles reported here present an attractive framework for the local or non-invasive delivery of DNA from the surfaces of implantable materials or biomedical devices.

Animals↗
PubMed2005

Surface analysis of erodible multilayered polyelectrolyte films: nanometer-scale structure and erosion profiles.

Atomic force microscopy (AFM) and scanning electron microscopy (SEM) coupled with ellipsometry have been used to characterize the microscale and nanoscale structures of erodible multilayered films fabricated from degradable polyamine 1 and either sodium poly(styrene sulfonate) (SPS) or plasmid DNA. Striking differences were found in the topography, structures, and erosion profiles of these two materials upon incubation in PBS buffer at 37 degrees C. For films fabricated from SPS, AFM data are consistent with an erosion process that occurs uniformly without the generation of holes or pits over large, micrometer-scale areas. By contrast, films fabricated from plasmid DNA undergo structural rearrangements to present surface-bound particles ranging in size from 50 to 400 nm. Additional characterization of these particulate structures by SEM suggested that they are interpenetrated with or fused to underlying polyelectrolyte layers on the silicon surface, providing a potential mechanism to manipulate the adhesive forces with which these particles are bound to the surface. The erosion profile observed for polymer 1/SPS films suggests that it may be possible to design assemblies that release two film components with well-defined release kinetics. In the context of gene delivery, the presentation of condensed DNA as nanoparticles at these surfaces may be advantageous with respect to stimulating the internalization and processing of DNA by cells. A quantitative understanding of the factors influencing the fabrication, structure, and erosion profiles of these materials will be useful for the design of multilayered assemblies for specific applications in which controlled film erosion or the release of therapeutic materials is desired.

Kinetics↗
PubMed2005

Tunable drug release from hydrolytically degradable layer-by-layer thin films.

The development of new thin film fabrication techniques that allow for precise control of degradation and drug release properties could represent an important advance in the fields of drug delivery and biomedicine. Polyelectrolyte layer-by-layer (LBL) thin films can be assembled with nanometer scale control over spatial architecture and morphology, yet very little work has focused on the deconstruction of these ordered thin films for controlled release applications. In this study, hydrolytically degradable LBL thin films are constructed by alternately depositing a degradable poly(beta-amino ester) (polymer 1) and a series of model therapeutic polysaccharides (heparin, low molecular weight heparin, and chondroitin sulfate). These films exhibit pH-dependent, pseudo-first-order degradation and release behavior. The highly versatile and tunable properties of these materials make them exciting candidates for the controlled release of a wide spectrum of therapeutics.

Journal Article↗
PubMed2004

Multilayered thin films that sustain the release of functional DNA under physiological conditions.

The development of thin films and coatings that control the release of DNA from the surfaces of materials could have a significant impact on localized approaches to gene therapy. Here, we report multilayered polyelectrolyte assemblies that sustain the release of functional plasmid DNA from the surfaces of model substrates under physiological conditions. Multilayered assemblies consisting of alternating layers of plasmid DNA encoding for enhanced green fluorescent protein (EGFP) and a synthetic degradable polyamine were deposited on planar silicon and quartz substrates using a layer-by-layer fabrication process. Film growth was monitored by ellipsometry and UV spectrophotometry and correlated linearly with the number of polymer and plasmid layers deposited. In general, the thickness of deposited layers was found to be a function of both the pH and the ionic strength of the polyelectrolyte solutions used. Films up to 100 nm thick were investigated in this study. These assemblies erode gradually upon incubation in phosphate-buffered saline at 37 degrees C, as determined by ellipsometry and UV spectrophotometry, and sustain the release of incorporated plasmid into the incubation medium for a period of up to 30 h. Characterization of the released plasmid by agarose gel electrophoresis revealed that the DNA was released in a relaxed, open circular, rather than supercoiled, topology; subsequent cell transfection experiments demonstrated that the released plasmid is transcriptionally viable and promotes the expression of EGFP in the COS-7 cell line. These layered materials could represent an approach to the controlled administration of one or more functional DNA constructs from the surfaces of biomedical materials and devices.

Animals↗
PubMed2004

Poly-beta amino ester-containing microparticles enhance the activity of nonviral genetic vaccines.

Current nonviral genetic vaccine systems are less effective than viral vaccines, particularly in cancer systems where epitopes can be weakly immunogenic and antigen-presenting cell processing and presentation to T cells is down-regulated. A promising nonviral delivery method for genetic vaccines involves microencapsulation of antigen-encoding DNA, because such particles protect plasmid payloads and target them to phagocytic antigen-presenting cells. However, conventional microparticle formulations composed of poly lactic-co-glycolic acid take too long to release encapsulated payload and fail to induce high levels of target gene expression. Here, we describe a microparticle-based DNA delivery system composed of a degradable, pH-sensitive poly-beta amino ester and poly lactic-co-glycolic acid. These formulations generate an increase of 3-5 orders of magnitude in transfection efficiency and are potent activators of dendritic cells in vitro. When used as vaccines in vivo, these microparticle formulations, unlike conventional formulations, induce antigen-specific rejection of transplanted syngenic tumor cells.

Animals↗
PubMed2004

Biocidal activity of polystyrenes that are cationic by virtue of protonation.

[structure: see text] Poly(1) kills bacteria (Gram-positive and -negative) and lyses human erythrocytes; this biocidal profile is similar to that of the peptide toxin mellitin. Poly(1) has antibacterial activity comparable to that of a potent derivative of the host defense peptide magainin II, but lacks magainin's selectivity for bacteria over erythrocytes. An analogous N-quaternized polymer, poly(3), is less biocidal than poly(1), suggesting that reversible N-protonation leads to greater biocidal activity than does irreversible N-quaternization.

Anti-Bacterial Agents↗
PubMed2004

Poly(beta-amino ester)s promote cellular uptake of heparin and cancer cell death.

Heparin/heparan sulfate-like glycosaminoglycans (HSGAGs) are involved in diverse cellular processes in the extracellular matrix (ECM). The biological effect of HSGAGs depends on disaccharide content and physiological location within the ECM. HSGAGs are also brought into cells during membrane transcytosis and growth factor signaling while protein bound. We sought to probe the impact of free HSGAGs within the cell by using heparin as a model HSGAG. A library of poly(beta-amino ester)s, which internalize DNA, was examined for the capacity of its members to internalize heparin. Fourteen polymers enabled heparin internalization. The most efficacious polymer reduced murine melanoma cell growth by 73%. No glycosaminoglycan was as efficacious as highly sulfated, full-length heparin. Internalized heparin likely interferes with transcription factor function and subsequently induces apoptotic cell death. Therefore, internalized heparin is a novel mechanism for inducing apoptosis of cancer cells.

Animals↗
PubMed2003

Parallel synthesis and biophysical characterization of a degradable polymer library for gene delivery.

We recently reported the parallel synthesis of 140 degradable poly(beta-amino esters) via the conjugate addition of 20 primary or secondary amine monomers to seven different diacrylate monomers. To explore possible structure/function relationships and further characterize this class of materials, we investigated the ability of each DNA-complexing polymer to overcome important cellular barriers to gene transfer. The majority of vectors were found to be uptake-limited, but complexes formed from polymers B14 and G5 displayed high levels of internalization relative to "naked" DNA (18x and 32x, respectively). Effective diameter and zeta potential measurements indicated that, in general, small particle size and positive surface charge led to higher internalization rates. Of the 10 DNA/polymer complexes with the highest uptake levels, all had effective diameters less than 250 nm and nine had positive zeta potentials. Lysosomal trafficking was investigated by measuring the pH environment of delivered DNA. Complexes prepared with polymers G5, G10, A13, B13, A14, and B14 were found to have near neutral pH measurements, suggesting that they were able to successfully avoid trafficking to acidic lysosomes. This work highlights the value of parallel synthesis and screening approaches for the discovery of new polymers for gene delivery and the elucidation of structure/function relationships for this important class of materials.

3T3 Cells↗
PubMed2003

Poly(ethylene oxide)-modified poly(beta-amino ester) nanoparticles as a pH-sensitive biodegradable system for paclitaxel delivery.

The main objective of this study was to develop and characterize a pH-sensitive biodegradable polymeric nanoparticulate system for tumor-selective paclitaxel delivery. A representative hydrophobic poly(beta-amino ester) (poly-1) was synthesized by conjugate addition of 4,4'-trimethyldipiperidine with 1,4-butanediol diacrylate. Poly-1 (M(n) 10,000 daltons) nanoparticles were prepared by the controlled solvent displacement method in an ethanol-water system in the presence of Pluronic) F-108, a poly(ethylene oxide) (PEO)-containing non-ionic surfactant. Control and PEO-modified nanoparticles were characterized by Coulter counter, scanning electron microscopy (SEM), zeta potential measurements, and electron spectroscopy for chemical analysis (ESCA). Polymer degradation studies were performed in phosphate-buffered saline (PBS, pH 7.4) at 37 degrees C. Paclitaxel loading capacities and efficiencies were determined and release studies were performed in Tween)-80 (0.1%, w/v)-containing PBS at 37 degrees C. Control and PEO-modified nanoparticles, labeled with rhodamine-123, were incubated with BT-20 cells to examine the uptake and cellular distribution as a function of time. PEO-modified nanoparticles with an average size of 100-150 nm and a positive surface charge of 37.0 mV were prepared. SEM analysis showed distinct smooth, spherical particles. The ether (-C-O-) peak of the C(1s) envelope in ESCA confirmed the surface presence of PEO chains. Polymer biodegradation studies showed that almost 85% of the starting material degraded after 6 days. The maximum paclitaxel loading efficiency attained was 97% at 1.0% (w/w) of the drug. Paclitaxel release studies showed that approximately 10% was released in the first 24 h, 80% after 3 days, and the entire content was released in approximately 5 days. After 1 h of incubation, a large fraction of the administered control and PEO-modified poly-1 nanoparticles was internalized in BT-20 cells. Results of this study demonstrate that PEO-modified poly-1 nanoparticles could provide increased therapeutic benefit by delivering the encapsulated drug to solid tumors.

Drug Delivery Systems↗
PubMed2002

Construction of hydrolytically-degradable thin films via layer-by-layer deposition of degradable polyelectrolytes.

Hydrolytically degradable, multilayered films ranging from 10 to 100 nm have been constructed by the layer-by-layer deposition of degradable polycations and oppositely charged polyanions. Polycations play dual roles in these systems, serving as structural components of the film as well as transient elements designed to trigger release; polyanions serve as structural components and as entities to be released or delivered. The films erode in a controlled manner under physiological conditions and are suitable for the incorporation and subsequent controlled release of functional polyanions such as DNA.

Animals↗