the-submerged-printing-cells-onto-modified-surface-using-continuous

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

WASATCH MICROFLUIDICS

The bio-bar code assay is an assay for ultrasensitive detection of proteins. The main technical hurdle in bio-bar code assay development is achieving a dose-dependent, reproducible signal with low background. We report on a magnetic bead ELISA screening mechanism for characterizing antibody pairs that are effective for use in the bio-bar code assay. The normal isoform of prion protein was utilized as the target protein as dozens of antibodies have been developed against it. The development of an ultrasensitive assay for the detection of the various isoforms of PrP has the potential to enable significant advances in the diagnosis and understanding of transmissible spongiform encephalopathies, including transmission mechanisms, disease pathology, and potential therapeutics. With prion protein as the target, the magnetic bead ELISA identified pairs with high background and low signal in the bio-bar code assay. The magnetic bead ELISA was effective as a screening mechanism because it reduced assay time and cost and allowed for understanding of pair characteristics such as development times and signal-to-noise ratios.

Efficient screening of high-signal and low-background antibody pairs in the bio-bar code assay using prion protein as the target.

Although spider silk displays an amazing combination of strength and extensibility unrivalled by most synthetic biomaterials, its molecular architecture is relatively simplistic. Four primary amino acid block motifs (An, (GA)n, GPGXX, GGX) have been correlated with mechanical functions. Recent genetic engineering to control the mechanical behavior of synthetic silk fibers has verified much of the proposed structure/function relationship; however, the genetically defined exchange between strength and elasticity has proven not to be a direct relationship. Thus, complete control over the mechanical properties of a synthetic spider silk based fiber continues to elude scientists. The yet undefined factor(s) may be an element of the fabrication process. Natural silk production results from a combination of dehydration and protein alignment that occurs during concurrent spin and draw processes. While synthetic fiber production attempts to mimic 1) dehydration with a series of coagulating solvents and 2) protein alignment through the controlled extrusion of a concentration dependent spinning solution, the spinning and drawing processes are separated and occur sequentially. Many studies have been conducted which have examined multiple parameters; however, the spinning conditions which produce consistent mechanical properties, necessary for the progression toward any medical, commercial or military application, have not been identified. Here, we report on mathematical methods based on data from a variety of spinning conditions to characterize different impacting properties as either primary (i.e. a condition which directs or dictates mechanical properties of an individual fiber) or ptimizing (i.e. a condition which increases the engineered properties of the silk).

Analyzing the clustering effects of major ampullate silk mechanical properties - biomed 2009.

To address persistent 1-3% infection rates associated with orthopedic implant surgeries, the next generation of bone graft filler materials will no longer pharmacologically silent being endowed as a local drug delivery vehicle to maintain locally high levels of antibiotic. Bone allograft material, used as a structural support to fill the avascular spaces in bone defects, revision surgeries, and traumatic injury, can be used as a drug depot to provide effective antibiotic delivery over the orthopedically relevant six-to-eight week time period. Passive antibiotic coatings, applied in the surgical theater, are quickly depleted from the site, inadvertently promoting the development of drug-resistance. Alternatively, many promising controlled-delivery strategies provide an initial burst release of antibiotic within 24 to 72 hours; however, this remains inadequate to combat the onslaught of ubiquitous pathogens that can persist only to reemerge once drug concentrations fall below the minimal inhibitory concentration (MIC). To improve the longevity of this strategy, a variety of coating techniques were evaluated in which clinically-accepted, FDA-recognized, degradable polycaprolactone (PCL) polymer acts as a rate-controlling membrane to retard the release of the antibiotic tobramycin from allograft bone. Using a combination of dipping and rapid drying, the drug-releasing polymer coating was applied concurrently maintaining the high surface area of the allograft bone; however, SEM imaging reveled an imperfect coating that negatively affected the release kinetics. Altering the drug-containing polymer formulation to incorporate water provided a smoother, more uniform coat and ultimately improved the drug-release profile and longevity out to 5 weeks using both bacteriostatic and bacteriocidal assays. Additionally, drug bioactivity was assessed and confirmed between 2 and 4 weeks in the absence of the water-containing polymer.

Evaluating antibiotic release profiles as a function of polymer coating formulation - biomed 2011.

Poly(amidoamine) (PAMAM) dendrimers have been proposed for a variety of biomedical applications and are increasingly studied as model nanomaterials for such use. The dendritic structure features both modular synthetic control of molecular size and shape and presentation of multiple equivalent terminal groups. These properties make PAMAM dendrimers highly functionalizable, versatile single-molecule nanoparticles with a high degree of consistency and low polydispersity. Recent nanotoxicological studies showed that intravenous administration of amine-terminated PAMAM dendrimers to mice was lethal, causing a disseminated intravascular coagulation-like condition. To elucidate the mechanisms underlying this coagulopathy, in vitro assessments of platelet functions in contact with PAMAM dendrimers were undertaken. This study demonstrates that cationic G7 PAMAM dendrimers activate platelets and dramatically alter their morphology. These changes to platelet morphology and activation state substantially altered platelet function, including increased aggregation and adherence to surfaces. Surprisingly, dendrimer exposure also attenuated platelet-dependent thrombin generation, indicating that not all platelet functions remained intact. These findings provide additional insight into PAMAM dendrimer effects on blood components and underscore the necessity for further research on the effects and mechanisms of PAMAM-specific and general nanoparticle toxicity in blood.

Cationic PAMAM dendrimers disrupt key platelet functions.

Poly(amidoamine) (PAMAM) dendrimers are increasingly studied as model nanoparticles for a variety of biomedical applications, notably in systemic administrations. However, with respect to blood-contacting applications, amine-terminated dendrimers have recently been shown to activate platelets and cause a fatal, disseminated intravascular coagulation (DIC)-like condition in mice and rats. We here demonstrate that, upon addition to blood, cationic G7 PAMAM dendrimers induce fibrinogen aggregation, which may contribute to the in vivo DIC-like phenomenon. We demonstrate that amine-terminated dendrimers act directly on fibrinogen in a thrombin-independent manner to generate dense, high-molecular-weight fibrinogen aggregates with minimal fibrin fibril formation. In addition, we hypothesize this clot-like behavior is likely mediated by electrostatic interactions between the densely charged cationic dendrimer surface and negatively charged fibrinogen domains. Interestingly, cationic dendrimers also induced aggregation of albumin, suggesting that many negatively charged blood proteins may be affected by cationic dendrimers. To investigate this further, zebrafish embryos were employed to more specifically determine the speed of this phenomenon and the pathway- and dose-dependency of the resulting vascular occlusion phenotype. These novel findings show that G7 PAMAM dendrimers significantly and adversely impact many blood components to produce rapid coagulation and strongly suggest that these effects are independent of classic coagulation mechanisms. These results also strongly suggest the need to fully characterize amine-terminated PAMAM dendrimers in regard to their adverse effects on both coagulation and platelets, which may contribute to blood toxicity.

Cationic PAMAM dendrimers aggressively initiate blood clot formation.

Baxter received reports of visible precipitate, identified as calcium carbonate, forming during hemofiltration with Accusol 35 solution. Aim: To evaluate the potential for acute cardiopulmonary adverse effects of Accusol 35 containing exaggerated calcium carbonate particles.

A comparison of the potential for acute cardiopulmonary adverse effects in dogs during continuous veno-venous hemofiltration with accusol 35 solution with and without induced calcium carbonate particles.

The pharmaceutical industry is experiencing comeback sales growth due in large part to the industry's R&D efforts that center on biologics drug development. To facilitate that effort, tools are being developed for more effective biologic drug development. At the forefront of this effort is epitope characterization, in particular epitope binning, primarily due to the role an epitope plays in drug function. Here we detail the financial advantages of epitope binning including (1) increased R&D productivity due to increased work in process, (2) reduced number of "dead-end"candidates, and (3) increasedability to reengineer antibodies based on the epitope. With the arrival of array Surface Plasmon Resonance, this manuscript serves as a call to push epitope binning earlier in the biological drug discovery process.

The Importance of Epitope Binning for Biological Drug Discovery.

Infection remains a significant problem associated with biomedical implants and orthopedic surgeries, especially in revision total joint replacements. Recent advances in antibiotic-releasing bone void fillers (BVF) provide new opportunities to address these types of device-related orthopedic infections that often lead to substantial economic burdens and reduced quality of life. We report improvements made in fabrication and scalability of an antibiotic-releasing polycaprolactone-calcium carbonate/phosphate ceramic composite BVF using a new solvent-free, molten-cast fabrication process. This strategy provides the ability to tailor drug release kinetics from the BVF composite based on modifications of the inorganic substrate and/or the polymeric component, allowing extended tobramycin release at bactericidal concentrations. The mechanical properties of the new BVF composite are comparable to many reported BVFs and validate the relative homogeneity of fabrication. Most importantly, fabrication quality controls are correlated with favorable drug release kinetics, providing bactericidal activity to 10 weeks in vitro when the polycaprolactone component exceeds 98% w/w of the total polymer fraction. Furthermore, in a time kill study, tobramycin-releasing composite fragments inhibited S. aureus growth over 48 h at inoculums as high as 10(9) CFU/mL. This customizable antibiotic-releasing BVF polymer-inorganic biomaterial should provide osseointegrative and osteoconductive properties while contributing antimicrobial protection to orthopedic sites requiring the use of bone void fillers. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.

Molded polymer-coated composite bone void filler improves tobramycin controlled release kinetics.

Of the diverse analytical tools used in proteomics, protein microarrays possess the greatest potential for providing fundamental information on protein, ligand, analyte, receptor, and antibody affinity-based interactions, binding partners and high-throughput analysis. Microarrays have been used to develop tools for drug screening, disease diagnosis, biochemical pathway mapping, protein-protein interaction analysis, vaccine development, enzyme-substrate profiling, and immuno-profiling. While the promise of the technology is intriguing, it is yet to be realized. Many challenges remain to be addressed to allow these methods to meet technical and research expectations, provide reliable assay answers, and to reliably diversify their capabilities. Critical issues include: (1) inconsistent printed microspot morphologies and uniformities, (2) low signal-to-noise ratios due to factors such as complex surface capture protocols, contamination, and static or no-flow mass transport conditions, (3) inconsistent quantification of captured signal due to spot uniformity issues, (4) non-optimal protocol conditions such as pH, temperature, drying that promote variability in assay kinetics, and lastly (5) poor protein (e.g., antibody) printing, storage, or shelf-life compatibility with common microarray assay fabrication methods, directly related to microarray protocols. Conventional printing approaches, including contact (e.g., quill and solid pin), non-contact (e.g., piezo and inkjet), microfluidics-based, microstamping, lithography, and cell-free protein expression microarrays, have all been used with varying degrees of success with figures of merit often defined arbitrarily without comparisons to standards, or analytical or fiduciary controls. Many microarray performance reports use bench top analyte preparations lacking real-world relevance, akin to "fishing in a barrel", for proof of concept and determinations of figures of merit. This review critiques current protein-based microarray preparation techniques commonly used for analytical and function-based proteomics and their effects on array-based assay performance.

Article	Year
Efficient screening of high-signal and low-background antibody pairs in the bio-bar code assay using prion protein as the target. Analytical biochemistry\| PubMed ID: 18694717	2008
Analyzing the clustering effects of major ampullate silk mechanical properties - biomed 2009. Biomedical sciences instrumentation\| PubMed ID: 19369768	2009
Evaluating antibiotic release profiles as a function of polymer coating formulation - biomed 2011. Biomedical sciences instrumentation\| PubMed ID: 21525595	2011
Cationic PAMAM dendrimers disrupt key platelet functions. Molecular pharmaceutics\| PubMed ID: 22497592	2012
Cationic PAMAM dendrimers aggressively initiate blood clot formation. ACS nano\| PubMed ID: 23062017	2012
A comparison of the potential for acute cardiopulmonary adverse effects in dogs during continuous veno-venous hemofiltration with accusol 35 solution with and without induced calcium carbona... Blood purification\| PubMed ID: 23989183	2013
The Importance of Epitope Binning for Biological Drug Discovery. Current drug discovery technologies\| PubMed ID: 24266537	2013
Molded polymer-coated composite bone void filler improves tobramycin controlled release kinetics. Journal of biomedical materials research. Part B, Applied biomaterials\| PubMed ID: 24376164	2013
A critical comparison of protein microarray fabrication technologies. The Analyst\| PubMed ID: 24479125	2014

Benjamin D. Brooks

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