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Biomedical subjects

Jennifer L West

Publications and source records attributed to Jennifer L West.

At least 19 recordsLinked to original sources
PubMed2006

Laser-scanning lithography (LSL) for the soft lithographic patterning of cell-adhesive self-assembled monolayers.

We report the development of laser-scanning lithography (LSL), which employs a laser-scanning confocal microscope to pattern photoresists that can be utilized, for example, in the fabrication of masters for use in soft lithography. This convenient technique provides even exposure across the entire view field and facilitates accurate alignment of successive photoresist exposures. Features on the scale of 3 microm have been achieved to date with a 10x objective (NA 0.45). Virtual masks, instructions for laser irradiation, were drawn using the Region of Interest (ROI) function of a Zeiss LSM 510 microscope. These regions were then exposed to a 458 nm argon laser for 32 micros (0.9 mW/microm(2)). Differential interference contrast (DIC) imaging was utilized with a non-destructive 514 nm argon laser as an immediate quality check of each exposure, to align successive exposures, and to reduce chromatic aberration between imaging and exposure. Developed masters were replica-molded with poly(dimethylsiloxane) (PDMS); these masters were then utilized for microcontact printing of cell-adhesive self-assembled monolayers (SAMs) to demonstrate the utility of this process. Initial studies confirmed that human dermal fibroblast adhesion and spreading were limited to cell-adhesive SAM areas. LSL is a rapid, flexible, and readily available technique that will accelerate master design and preparation; moreover, it can be applied to additional forms of photolithography and photopolymerization for studies in cell biology, biomaterials design and evaluation, materials science, and surface chemistry.

Alkanes↗
PubMed2006

Correlating nanoscale titania structure with toxicity: a cytotoxicity and inflammatory response study with human dermal fibroblasts and human lung epithelial cells.

Nanocrystalline titanium dioxide (nano-TiO(2)) is an important material used in commerce today. When designed appropriately it can generate reactive species (RS) quite efficiently, particularly under ultraviolet (UV) illumination; this feature is exploited in applications ranging from self-cleaning glass to low-cost solar cells. In this study, we characterize the toxicity of this important class of nanomaterials under ambient (e.g., no significant light illumination) conditions in cell culture. Only at relatively high concentrations (100 microg/ml) of nanoscale titania did we observe cytotoxicity and inflammation; these cellular responses exhibited classic dose-response behavior, and the effects increased with time of exposure. The extent to which nanoscale titania affected cellular behavior was not dependent on sample surface area in this study; smaller nanoparticlulate materials had effects comparable to larger nanoparticle materials. What did correlate strongly to cytotoxicity, however, was the phase composition of the nanoscale titania. Anatase TiO(2), for example, was 100 times more toxic than an equivalent sample of rutile TiO(2). The most cytotoxic nanoparticle samples were also the most effective at generating reactive oxygen species; ex vivo RS species generation under UV illumination correlated well with the observed biological response. These data suggest that nano-TiO(2) samples optimized for RS production in photocatalysis are also more likely to generate damaging RS species in cell culture. The result highlights the important role that ex vivo measures of RS production can play in developing screens for cytotoxicity.

Cell Line↗
PubMed2006

Metal nanoshells.

Metal nanoshells are a new class of nanoparticles with highly tunable optical properties. Metal nanoshells consist of a dielectric core nanoparticle such as silica surrounded by an ultrathin metal shell, often composed of gold for biomedical applications. Depending on the size and composition of each layer of the nanoshell, particles can be designed to either absorb or scatter light over much of the visible and infrared regions of the electromagnetic spectrum, including the near infrared region where penetration of light through tissue is maximal. These particles are also effective substrates for surface-enhanced Raman scattering (SERS) and are easily conjugated to antibodies and other biomolecules. One can envision a myriad of potential applications of such tunable particles. Several potential biomedical applications are under development, including immunoassays, modulated drug delivery, photothermal cancer therapy, and imaging contrast agents.

Animals↗
PubMed2006

Angiogenesis-like activity of endothelial cells co-cultured with VEGF-producing smooth muscle cells.

A number of strategies have been investigated to improve therapeutic vascularization of ischemic and bioengineered tissues. In these studies, we genetically modified vascular smooth muscle cells (VSMC) to promote endothelial cell proliferation, migration, and formation of microvascular networks. VSMCs were virally transduced to produce vascular endothelial growth factor (VEGF), which acts as a chemoattractant and mitogen of endothelial cells (EC). VSMCs transduced with VEGF(165) cDNA produced significant levels of the protein (2-4 ng/10(5) cell/day). The proliferation of ECs increased after exposure to VEGF-transfected SMCs or their conditioned media. The chemotactic response of ECs to the VEGF-producing cells was explored in two in vitro systems, the modified Boyden chamber assay and a 2-D fence-style migration assay, and both demonstrated increased migration of ECs in response to VEGF-transfected cells. Furthermore, endothelial cells seeded on top of the VEGF-transfected SMCs formed capillary-like structures. These results suggest that VSMCs genetically modified to produce VEGF could be a potential delivery mechanism to enhance endothelial cell migration and subsequent capillary formation, which in turn could improve vascularization of ischemic or regenerating tissue. Furthermore, this system could potentially be used as an in vitro test bed for evaluation of novel angiogenic and anti-angiogenic compounds.

Animals↗
PubMed2006

Novel heparanase-inhibiting antibody reduces neointima formation.

Basic fibroblast growth factor (bFGF), stored bound to heparan sulfate proteoglycans in the extracellular matrix (ECM) of the arterial media, may initiate smooth muscle cell (SMC) proliferation after coronary intervention, thus contributing to restenosis. bFGF mobilization from ECM stores after injury may be induced by platelet degranulation products such as heparanase. Therapies aimed at the inhibition of bFGF release and activation may assist in prevention of restenosis. To test this theory, we first examined the mobilization and activation of bFGF in the arterial media by platelet-derived heparanase. Heparanase, locally delivered to the rat carotid artery, was found to release bFGF and induce substantial SMC proliferation in the absence of actual vascular injury. An antibody that neutralizes heparanase was then developed and evaluated in a rat carotid balloon injury model. Local delivery of anti-heparanase IgG was found to inhibit bFGF release by approximately 60% ( p < 0.001) at 4 d; this correlated with the significant reduction in neointima formation observed at 14 d (intimal area/medial area: control 1.3 +/- 0.3, anti-heparanase 0.35 +/- 0.12, p < 0.0001). Platelet-derived heparanase is therefore likely to be important in initiating events leading to restenosis via bFGF mobilization. Furthermore, heparanase neutralization may assist in the prevention of restenosis following vascular injury.

Animals↗
PubMed2005

Photolithographic patterning of polyethylene glycol hydrogels.

A simple, inexpensive photolithographic method for surface patterning deformable, solvated substrates is demonstrated using photoactive poly(ethylene glycol) (PEG)-diacrylate hydrogels as model substrates. Photolithographic masks were prepared by printing the desired patterns onto transparencies using a laser jet printer. Precursor solutions containing monoacryloyl-PEG-peptide and photoinitiator were layered onto hydrogel surfaces. The acrylated moieties in the precursor solution were then conjugated in monolayers to specific hydrogel regions by exposure to UV light through the transparency mask. The effects of UV irradiation time and precursor solution concentration on the levels of immobilized peptide were characterized, demonstrating that bound peptide concentration can be controlled by tuning these parameters. Multiple peptides can be immobilized to a single hydrogel surface in distinct patterns by sequential application of this technique, opening up its potential use in co-cultures. In addition, 3D structures can be generated by incorporating PEG-diacrylate into the precursor solution. To evaluate the feasibility of using these patterned surfaces for guiding cell behavior, human dermal fibroblast adhesion on hydrogel surfaces patterned with acryloyl-PEG-RGDS was investigated. This patterning method may find use in tissue engineering, the elucidation of fundamental structure-function relationships, and the formation of immobilized cell and protein arrays for biotechnology.

Cells, Cultured↗
PubMed2005

Covalent immobilization of RGDS on hydrogel surfaces to direct cell alignment and migration.

This study extends the capability for directing cell behavior using PEG-based hydrogels in tissue-engineering applications to include control over the spatial distribution of the adhesive peptide, RGDS. A continuous linear gradient was formed by simultaneously using a gradient maker to combine precursor solutions and using photopolymerization to lock the RGDS gradient in place. Hydrogels containing entrapped gradients of bovine serum albumin (BSA) were characterized using Coomassie brilliant blue stain, which indicated that BSA concentration increases along the hydrogel's length and that the steepness of the gradient's slope can be varied by changing the relative BSA concentrations in the precursor solutions. Human dermal fibroblasts responded to covalently immobilized RGDS gradients by changing their morphology to align in the direction of increasing RGDS concentration. After 24 h, approximately 46% of fibroblasts were aligned with the RGDS-gradient axis. This proportion of cells further increased to approximately 53% (p < 0.05) and approximately 58% after 48 and 96 h, respectively. Also, fibroblasts migrated differentially depending on the concentration of RGDS. Fibroblasts migrated approximately 48% further going up the concentration gradient (0 to 6 micromol/ml PEG-RGDS) than going down the concentration gradient. Migration up the concentration gradient was also approximately 33% greater than migration on control surfaces with a constant concentration of RGDS (2 micromol/ml), while migration down the gradient was reduced approximately 12% relative to the control surface. In addition, directed migration was further enhanced by increasing the RGDS gradient's slope. This hydrogel system is expected to be useful for directing cell migration to enhance the formation of engineered tissues.

Cell Movement↗
PubMed2005

Functionalization density dependence of single-walled carbon nanotubes cytotoxicity in vitro.

The cytotoxic response of cells in culture is dependant on the degree of functionalization of the single-walled carbon nanotube (SWNT). After characterizing a set of water-dispersible SWNTs, we performed in vitro cytotoxicity screens on cultured human dermal fibroblasts (HDF). The SWNT samples used in this exposure include SWNT-phenyl-SO(3)H and SWNT-phenyl-SO(3)Na (six samples with carbon/-phenyl-SO(3)X ratios of 18, 41, and 80), SWNT-phenyl-(COOH)(2) (one sample with carbon/-phenyl-(COOH)(2) ratio of 23), and underivatized SWNT stabilized in 1% Pluronic F108. We have found that as the degree of sidewall functionalization increases, the SWNT sample becomes less cytotoxic. Further, sidewall functionalized SWNT samples are substantially less cytotoxic than surfactant stabilized SWNTs. Even though cell death did not exceed 50% for cells dosed with sidewall functionalized SWNTs, optical and atomic force microscopies show direct contact between cellular membranes and water-dispersible SWNTs; i.e. the SWNTs in aqueous suspension precipitate out and selectively deposit on the membrane.

Cells, Cultured↗
PubMed2005

Localized delivery of nitric oxide from hydrogels inhibits neointima formation in a rat carotid balloon injury model.

Using novel nitric oxide (NO)-generating polymeric hydrogels that can be rapidly photopolymerized in situ, we can deliver NO locally at the site of vascular injury. Depending on material design, these poly(ethylene glycol) (PEG)-based hydrogels can generate NO for up to 50 d. This study demonstrates the ability of nitric oxide-generating hydrogels (PEG-Cys-NO) to influence key components of the restenosis cascade both in vitro and in vivo. PEG-Cys-NO hydrogels inhibited smooth muscle cell proliferation, increased endothelial cell proliferation, and inhibited platelet adhesion in vitro. Moreover, in vivo, PEG-Cys-NO hydrogels inhibited intimal thickening in a rat carotid balloon injury model. The perivascular application of NO-generating polymers post-injury reduced neointima formation at 14 d by approximately 80% compared to controls (intimal area/medial area (I/M): PEG-Cys-NO=0.20+/-0.17, control=0.84+/-0.19, p<0.00002; intimal thickness: PEG-Cys-NO=12+/-10 microm, control=60+/-18 microm, p<0.00002). Treatment with the PEG-Cys-NO hydrogels caused a significant decrease in the per cent of proliferating cell nuclear antigen positive medial cells (29+/-5%) at 4 d as compared to treatment with the control hydrogels (51+/-1%, p<0.02). Additionally, vessel re-endothelialization at 14 d was slightly enhanced in the presence of the NO-generating hydrogels. These data indicate that localized delivery of NO from these hydrogels can significantly inhibit neointima formation in a rat carotid balloon injury model and suggest that these materials may be useful in preventing restenosis.

Angioplasty, Balloon, Coronary↗
PubMed2005

Protease-activated quantum dot probes.

We have developed a novel nanoparticulate luminescent probe with inherent signal amplification upon interaction with a targeted proteolytic enzyme. This construct may be useful for imaging in cancer detection and diagnosis. In this system, quantum dots (QDs) are bound to gold nanoparticles (AuNPs) via a proteolytically degradable peptide sequence to non-radiatively suppress luminescence. A 71% reduction in luminescence was achieved with conjugation of AuNPs to QDs. Release of AuNPs by peptide cleavage restores radiative QD photoluminescence. Initial studies observed a 52% rise in luminescence over 47 h of exposure to 0.2 mg/mL collagenase. These probes can be customized for targeted degradation simply by changing the sequence of the peptide linker.

Coated Materials, Biocompatible↗
PubMed2005

Modification of polyurethaneurea with PEG and YIGSR peptide to enhance endothelialization without platelet adhesion.

Improved endothelialization without platelet adhesion is essential to enhance the long-term patency of synthetic vascular grafts and other blood-contacting devices. We have developed a dually modified polyurethaneurea by incorporating endothelial cell adhesive YIGSR peptide sequences as chain extenders and nonthrombogenic PEG as a soft segment (PUUYIGSR-PEG) in the polymer backbone. PUUYIGSR-PEG was successfully synthesized and characterized by proton NMR, FTIR, GPC, DSC, ESCA, and contact angle measurement. Despite having similar molecular weight, the peptide/PEG-modified polyurethaneurea (PUUYIGSR-PEG) showed superior mechanical properties compared to the control PEG-modified polyurethaneurea (PUUPPD-PEG). Virtually no platelet adhesion was observed on PUUYIGSR-PEG, while endothelial cell adhesion, spreading, and migration were significantly greater on PUUYIGSR-PEG compared to PUUPPD-PEG. Thus, this bioactive polymer may be an appropriate biomaterial for small diameter vascular grafts.

Amino Acid Sequence↗
PubMed2005

Near infrared laser-tissue welding using nanoshells as an exogenous absorber.

BACKGROUND AND OBJECTIVE: Gold nanoshells are a new class of nanoparticles that can be designed to strongly absorb light in the near infrared (NIR). These particles provide much larger absorption cross-sections and efficiency than can be achieved with currently used chemical chromophores without photobleaching. In these studies, we have investigated the use of gold nanoshells as exogenous NIR absorbers to facilitate NIR laser-tissue welding. STUDY DESIGN/MATERIALS AND METHODS: Gold nanoshells with peak extinction matching the NIR wavelength of the laser being used were manufactured and suspended in an albumin solder. Optimization work was performed on ex vivo muscle samples and then translated into testing in an in vivo rat skin wound-healing model. Mechanical testing of the muscle samples was immediately performed and compared to intact tissue mechanical properties. In the in vivo study, full thickness incisions in the dorsal skin of rats were welded, and samples of skin were excised at 0, 5, 10, 21, and 32 days for analysis of strength and wound healing response. RESULTS: Mechanical testing of nanoshell-solder welds in muscle revealed successful fusion of tissues with tensile strengths of the weld site equal to the uncut tissue. No welding was accomplished with this light source when using solder formulations without nanoshells. Mechanical testing of the skin wounds showed sufficient strength for closure and strength increased over time. Histological examination showed good wound-healing response in the soldered skin. CONCLUSIONS: The use of nanoshells as an exogenous absorber allows the usage of light sources that are minimally absorbed by tissue components, thereby, minimizing damage to surrounding tissue and allowing welding of thicker tissues.

Animals↗
PubMed2005

Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration.

Basic fibroblast growth factor (bFGF) was immobilized to hydrogel scaffolds with retention of mitogenic and chemotactic activity. The bFGF was functionalized in order to incorporate it covalently within polyethylene glycol (PEG) hydrogel scaffolds by reaction with acryloyl-PEG-NHS. Hydrogels were formed by exposing aqueous solutions of PEG diacrylate, acryloyl-PEG-RGDS, and acryloyl-PEG-bFGF to long-wavelength ultraviolet light in the presence of a photoinitiator. These bFGF-modified hydrogels with RGD adhesion sites were evaluated for their effect on vascular smooth muscle cell (SMC) behavior, increasing SMC proliferation by approximately 41% and migration by approximately 15%. A covalently immobilized bFGF gradient was formed using a gradient maker to pour the hydrogel precursor solutions and then photopolymerizing to lock in the concentration gradient. Silver staining was used to detect the bFGF gradient, which increased linearly along the hydrogel's length. Cells were observed to align on hydrogels modified with a bFGF gradient in the direction of increasing tethered bFGF concentration as early as 24 h after seeding. SMCs also migrated differentially, up the concentration gradient, on bFGF-gradient hydrogels compared to control hydrogels with and without a constant bFGF concentration. These hydrogel scaffolds may be useful for studying protein gradient effects on cell behavior and for directing cell migration in tissue-engineering applications.

Aorta↗
PubMed2005

Nano-C60 cytotoxicity is due to lipid peroxidation.

This study examines the biological effects of water-soluble fullerene aggregates in an effort to evaluate the fundamental mechanisms that contribute to the cytotoxicity of a classic engineered nanomaterial. For this work we used a water-soluble fullerene species, nano-C60, a fullerene aggregate that readily forms when pristine C60 is added to water. Nano-C60 was cytotoxic to human dermal fibroblasts, human liver carcinoma cells (HepG2), and neuronal human astrocytes at doses>or= 50 ppb (LC50=2-50 ppb, depending on cell type) after 48 h exposure. This water-soluble nano-C60 colloidal suspension disrupts normal cellular function through lipid peroxidation; reactive oxygen species are responsible for the membrane damage. Cellular viability was determined through live/dead staining and LDH release. DNA concentration and mitochondrial activity were not affected by the nano-C60 inoculations to cells in culture. The integrity of cellular membrane was examined by monitoring the peroxy-radicals on the lipid bilayer. Subsequently, glutathione production was measured to assess the cell's reaction to membrane oxidation. The damage to cell membranes was observed both with chemical assays, and confirmed physically by visualizing membrane permeability with high molecular weight dyes. With the addition of an antioxidant, L-ascorbic acid, the oxidative damage and resultant toxicity of nano-C60 was completely prevented.

Antioxidants↗
PubMed2005

Nitric oxide-generating hydrogels inhibit neointima formation.

This study evaluated the effects of localized delivery of nitric oxide (NO) from hydrogels covalently modified with S-nitrosocysteine (Cys-NO) on neoinitma formation, a key component of restenosis, in a rat balloon-injury model. Soluble Cys-NO was used in preliminary studies to identify dosage ranges that were able to simultaneously inhibit smooth muscle cell proliferation, enhance endothelial cell proliferation, and reduce platelet adhesion. Photo-cross-linked PEG-based hydrogels were formed with covalently immobilized Cys-NO. These materials release NO for approximately 24 h and can be applied to tissues and photo-cross-linked in situ to form local drug-delivery systems. Localized delivery of NO from hydrogels containing Cys-NO inhibited neointima formation in a rat balloon-injury model by approximately 75% at 14 days.

Angioplasty, Balloon, Coronary↗
PubMed2005

Whole-blood immunoassay facilitated by gold nanoshell-conjugate antibodies.

In this chapter, we outline a simple procedure using gold nanoshells as a substrate for an immunoassay that is capable of detecting subnanogram levels of analyte within whole blood on the order of minutes. Unique to metallic nanoshells is their optical tunability over a large range of wavelengths. We describe the design of nanoshells that attenuate light strongly in a region of light where blood does not (i.e., the near infrared [IR]), permitting optical detection of nanoshells in whole blood. We also describe a procedure to monitor the analyte-induced aggregation of antibody-conjugated nanoshells in whole blood using near-IR light. The immunoassay is fast and specific, requires no separation/purification steps, is simple to perform (mix and sit), and uses common laboratory equipment for detection (spectrophotometer). Preparation of the antibody-nanoshell conjugates is described, along with the design and optimization of the whole-blood nanoshell-based immunoassay system.

Animals↗
PubMed2004

Photo-thermal tumor ablation in mice using near infrared-absorbing nanoparticles.

The following study examines the feasibility of nanoshell-assisted photo-thermal therapy (NAPT). This technique takes advantage of the strong near infrared (NIR) absorption of nanoshells, a new class of gold nanoparticles with tunable optical absorptivities that can undergo passive extravasation from the abnormal tumor vasculature due to their nanoscale size. Tumors were grown in immune-competent mice by subcutaneous injection of murine colon carcinoma cells (CT26.WT). Polyethylene glycol (PEG) coated nanoshells (approximately 130 nm diameter) with peak optical absorption in the NIR were intravenously injected and allowed to circulate for 6 h. Tumors were then illuminated with a diode laser (808 nm, 4 W/cm2, 3 min). All such treated tumors abated and treated mice appeared healthy and tumor free >90 days later. Control animals and additional sham-treatment animals (laser treatment without nanoshell injection) were euthanized when tumors grew to a predetermined size, which occurred 6-19 days post-treatment. This simple, non-invasive procedure shows great promise as a technique for selective photo-thermal tumor ablation.

Animals↗