W. Todd Monroe

Scarless skin repair in immunodeficient mice

Scarring, the end result of the wound healing process in adult mammals, is a problem of significant clinical importance. We observed that athymic nude‐nu mice, similar to mammalian fetuses, are able to restore the structure and integrity of injured skin through a process resembling regeneration, where scar formation is absent. Among the postinjured skin tissues collected from athymic nude‐nu, wild‐type controls (C57BL/6J), severe‐combined immunodeficient, Rag (lack of B and T cells), athymic (thymectomized neonates and adult C57BL/6J), and mice treated with an immunosuppressant (cyclosporin A), only athymic nude‐nu mice showed: a lack of scar by histological examination (hematoxylin & eosin and Massons trichrome staining), low levels of collagen (as determined by hydroxyproline content), high levels of hyaluronic acid, a statistically significant increase in elastic modulus for injured samples over unwounded (biomechanical testing) and low levels of the pro‐scarring cytokines platelet‐derived growth factor‐B and transforming growth factor β1. Additionally, immunohistochemical and Western blot analyses of postinjured tissues as well as flow cytometry analysis of blood samples showed the presence of CD8‐positive cells in all studied animals except nude‐nu mice. We conclude that scarless skin healing in athymic nude‐nu mice provides a new model to study the influence of the immune system on tissue regeneration.

Caged siRNAs for spatiotemporal control of gene silencing.

Various strategies have been employed to achieve control over delivery of siRNA molecules to intended target cells. Photocaging is one specific class of modifications for silencing oligonucleotides that block their bioactivity until exposure to near-ultraviolet light. These caged RNAi effectors enable both spatial and temporal targeting of a dosed release of gene silencing agents by directed light exposure that photocleaves the cage moieties. Herein we compare the photochemical properties of cage compounds and strategies for their use, attached either randomly or site-specifically, to demonstrate various forms of gene expression regulation in vitro and in vivo. This light-controllable strategy has potential applications for precisely probing developing biological systems and eventually enabling targeted gene-silencing therapeutics.

Silver Nanoscale Antisense Drug Delivery System for Photoactivated Gene Silencing

The unique photophysical properties of noble metal nanoparticles contribute to their potential as photoactivated drug delivery vectors. Here we demonstrate the synthesis and characterization of 60-80 nm silver nanoparticles (SNPs) decorated with thiol-terminated photolabile DNA oligonucleotides. In vitro assays and fluorescent confocal microscopy of treated cell cultures show efficient UV-wavelength photoactivation of surface-tethered caged ISIS2302 antisense oligonucleotides possessing internal photocleavable linkers. As a demonstration of the advantages of these novel nanocarriers, we investigate properties including: enhanced stability to nucleases, increased hybridization activity upon photorelease, and efficient cellular uptake as compared to commercial transfection vectors. Their potential as multicomponent delivery agents for oligonucleotide therapeutics is shown through regulation of ICAM-1 (Intracellular Adhesion Molecule-1) silencing. Our results suggest a means to achieve light-triggered, spatiotemporally controlled gene silencing via nontoxic silver nanocarriers, which hold promise as tailorable platforms for nanomedicine, gene expression studies, and genetic therapies.

Control of DNA Hybridization with Photocleavable Adducts

Previous reports have shown that 1‐(4,5‐dimethoxy‐2‐nitro‐phenyl)ethyl ester (DMNPE) adducts coupled to DNA plasmids block transcription in vitro and in vivo until removed with light. In this report, we explore the use of DMNPE to control DNA hybridization. We found that DMNPE‐caged oligonucleotides have changed spectrophotometric and elec‐trophoretic properties that can be restored with light exposure. Caged oligonucleotides have slower electrophoretic mobility than noncaged oligonucleotides and caged oligonucleotides exposed to light. Effects of caging on hybridization were assessed in a fluorescence‐based assay using a 20mer caged DNA oligonucleotide complementary to a 30mer molecular beacon. Fluorescence results indicate that hybridization is reduced and subsequently restored by light. Subsequent gel shift assays confirmed these results. Hybridization activity of caged oligonucleotides with an average of 14–16 DMNPE adducts per oligonucleotide was 14% of noncaged control oligonucleotides and after 365 nm photolysis, increased to nearly 80% of controls. Spectrophotometric characterization of caged oligonucleotides exposed to light and then filtered to remove the released DMNPE adducts indicates two to four attached cage groups remaining following photoactivation. These results suggest that this light‐based technology can be used as a tool for the spatial and temporal regulation of hybridization‐based DNA bioactivity.

Photobiological and thermal effects of photoactivating UVA light doses on cell cultures

While near-ultraviolet light has been widely used to photoactivate fluorophores and caged compounds in cells, little is known of the long-term biological effects of this light. UVA (315-400 nm) photoactivating light has been well characterized in short-term cell studies and is now being employed in higher doses to control longer-duration phenomena (e.g. gene expression). Annexin V-Cy5/propidium iodide apoptosis flow cytometry assays were used to determine responses of HeLa cells to doses of UVA light up to 23.85 J cm(-2). Cells seeded at low densities had higher percentages of apoptosis and necrosis and were also more susceptible to UVA damage than cells seeded at higher densities. The dose to induce apoptosis and death in 50% of the cells (dose(1/2)) was determined for two different commercially available UVA light sources: 7.6 J cm(-2) for the GreenSpot photocuring system and 2.52 J cm(-2) for the BlakRay lamp. All BlakRay doses tested had significant cellular responses, whereas no significant cellular responses were found for doses below 1.6 J cm(-2) from the GreenSpot light source. A temperature control and measurement system was used to determine direct heating from the UVA sources and also the effect that cooling cell cultures during photoexposure has on minimizing cell damage. Cooling during the BlakRay photoexposure significantly reduced the percentage of necrotic cells, but there was no significant difference for cooling during photoactivation with the GreenSpot. Differences in cell responses to similar UVA doses of different intensities suggest that photoduration should be considered along with total dose and thermal conditions in photoactivation studies.

Fully 2′-Deoxy-2′-Fluoro Substituted Nucleic Acids Induce RNA Interference in Mammalian Cell Culture

RNA interference is a phenomenon in which RNA molecules elicit potent and sequence‐specific post‐transcriptional gene silencing. Recent studies have shown that small interfering RNA containing pyrimidine 2′‐fluoro modifications elicit RNAi. In this study, we demonstrate that fully‐2′‐fluorinated nucleic acids can be generated for RNAi studies through either custom solid‐phase synthesis or in vitro transcription using a mutated polymerase and fluorinated nucleoside triphosphates. Single‐stranded and hybridized fully‐2′‐fluorinated nucleic acids were subjected to a ribonuclease to assess their resistance to digestion. Duplex siFNA and antisense fully‐2′‐fluorinated nucleic acids were evaluated for their ability to knockdown green fluorescent protein expression in mammalian cell culture. Based on the results, fully‐2′‐fluorinated nucleic acids can be successfully generated, and fully‐2′‐fluorinated nucleic acids products show superior resistance to digestion over native RNA. Melt curve analysis suggests that transcribed fully‐2′‐fluorinated nucleic acids may contain base miscoding errors or early termination products. Small interfering fluoronucleic acid can induce RNAi and the silencing efficiency is nearly equivalent to the unmodified small interfering RNA species. Silencing from antisense fully‐2′‐fluorinated nucleic acids was greatly reduced relative to the duplex form. The lack of silencing activity from single‐stranded fully‐2′‐fluorinated nucleic acids, combined with reverse transcription polymerase chain reaction data showing that mRNA decreases following siFNA treatment, suggests that knockdown from siFNA is likely enzymatically driven as opposed to simple translational arrest.

A Planar Microfluidic Mixer Based on Logarithmic Spirals

A passive, planar micromixer design based on logarithmic spirals is presented. The device was fabricated using polydimethylsiloxane soft photolithography techniques, and mixing performance was characterized via numerical simulation and fluorescent microscopy. Mixing efficiency initially declined as Reynolds number increased, and this trend continued until a Reynolds number of 15 where a minimum was reached at 53%. Mixing efficiency then began to increase reaching a maximum mixing efficiency of 86% at Re = 67. Three-dimensional simulations of fluid mixing in this design were compared to other planar geometries such as the Archimedes spiral and Meandering-S mixers. The implementation of logarithmic curvature offers several unique advantages that enhance mixing, namely a variable cross-sectional area and a logarithmically varying radius of curvature that creates 3-D Dean vortices. These flow phenomena were observed in simulations with multilayered fluid folding and validated with confocal microscopy. This design provides improved mixing performance over a broader range of Reynolds numbers than other reported planar mixers, all while avoiding external force fields, more complicated fabrication processes, and the introduction of flow obstructions or cavities that may unintentionally affect sensitive or particulate-containing samples. Due to the planar design requiring only single-step lithographic features, this compact geometry could be easily implemented into existing micro-total analysis systems requiring effective rapid mixing.

Photoactivated miR-148b–nanoparticle conjugates improve closure of critical size mouse calvarial defects

Inducible systems providing temporal control of differentiation have the potential to improve outcomes in surgical reconstruction and regenerative medicine by precise modulation of wound healing and tissue repair processes. The aim of this study was to demonstrate that nanoformulated microRNA (miRNA) conjugates activated via photo exposure can lead to the induced osteogenic differentiation of human adipose-derived stromal/stem cells (hASCs) in vivo. The conjugate PC-miR-148b-SNP, a mimic of miRNA-148b tethered to silver nanoparticles (SNPs) via a photolabile linker, was used to modulate gene expression for improved closure of a critical size defect drilled on the right parietal bone of male CD-1 nude homozygous mice. The PC-miR-148b-SNP conjugates added to hASCs and loaded to either Matrigel or polycaprolactone (PCL) scaffolds resulted in different levels of healing of the defect. After 4 and 12weeks, 3-D micro-computed tomography reconstructed images indicate statistically significant defect closure from 3.83±1.19% to 5.46±2.01% and 6.54±4.28% to 32.53±8.3% for non-photoactivated and photoactivated conjugates, respectively, in the PCL scaffolds. The results were confirmed with H&E and Massons Trichrome stains in the transverse sections of photoactivated conjugates. Collagen fiber staining was greatest at 12weeks when it reached approximately the same density and thickness as the native calvarium. This technology provides a platform that can be used with other miRNAs that actively govern the pathways responsible for regenerative and wound healing processes.

Antimicrobial biocompatible bioscaffolds for orthopaedic implants.

Nationally, nearly 1.5 million patients in the USA suffer from ailments requiring bone grafts and hip and other joint replacements. Infections following internal fixation in orthopaedic trauma can cause osteomyelitis in 22–66% of cases and, if uncontrolled, the mortality rate can be as high as 2%. We characterize a procedure for the synthesis of antimicrobial and biocompatible poly‐l‐lactic acid (PLLA) and poly‐ethyleneglycol (PEG) bioscaffolds designed to degrade and absorb at a controlled rate. The bioscaffold architecture aims to provide a suitable substrate for the controlled release of silver nanoparticles (SNPs) to reduce bacterial growth and to aid the proliferation of human adipose‐derived stem cells (hASCs) for tissue‐engineering applications. The fabricated bioscaffolds were characterized by scanning transmission microscope (SEM) and it showed that the addition of tncreasing concentrations of SNPs results in the formation of dendritic porous channels perpendicular to the axis of precipitation. The antimicrobial properties of these porous bioscaffolds were tested according to a modified ISO 22196 standard across varying concentrations of biomass‐mediated SNPs to determine an efficacious antimicrobial concentration. The bioscaffolds reduced the Staphylococcus aureus and Escherichia coli viable colony‐forming units by 98.85% and 99.9%, respectively, at an antimicrobial SNPs concentration of 2000 ppm. Human ASCs were seeded on bioscaffolds and cultured in vitro for 20 days to study the effect of SNPs concentration on the viability of cells. SEM analysis and the metabolic activity‐based fluorescent dye, AlamarBlue®, demonstrated the growth of cells on the efficacious antimicrobial bioscaffolds. The biocompatibility of in vitro leached silver, quantified by inductively coupled plasma optical emission spectroscopy (ICP‐OES), proved non‐cytotoxic when tested against hASCs, as evaluated by MTT assay. Copyright

Freezing and post-thaw apoptotic behaviour of cells in the presence of palmitoyl nanogold particles

The aim of this study was to evaluate the freezing response of HeLa and Jurkat cells in the presence of commercially available nanoparticles, NPs (Palmitoyl Nanogold®, Nanoprobes). The cells were incubated with NPs for either 5 min or 3 h, and a calorimeter technique was then used to generate the volumetric shrinkage response during freezing at 20 °C min−1. Concomitantly, we also examined the effect of a commonly used cryoprotectant, dimethylsulfoxide, DMSO (10% v/v ratio) on the freezing response of HeLa and Jurkat cells. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the reference hydraulic conductivity, Lpg, (μm/min-atm) and activation energy, ELp, (kcal mol−1) were obtained. For HeLa cells, the values of Lpg ranged from 0.08 to 0.23 µm/min-atm, while ELp ranged from 10.9 to 37.4 kcal mol−1. For Jurkat cells these parameter values ranged from 0.05 to 0.16 µm/min-atm and 9.5 to 35.9 kcal mol−1. A generic optimal cooling rate equation was then used to predict the optimal rates of freezing HeLa and Jurkat cells in the presence and absence of DMSO and NPs. The post-thaw viability and apoptotic response of HeLa and Jurkat cells was further investigated by cooling cells at three rates in the presence and absence of DMSO and NPs using a commercially available controlled rate freezer. Jurkat cells treated in this manner demonstrated an increase in their adhesive properties after 18 h incubation and adhered strongly to the bottom of the culture plate. This observation prevented further analysis of Jurkat apoptotic and necrotic post-thaw responses. There was no significant effect of NPs or DMSO alone on HeLa cell viability prior to freezing. The post-thaw results from HeLa cells show that the NPs increased the measured post-freeze apoptotic response when cooled at 1 °C min−1, suggesting a possible therapeutic use of NPs in cryodestructive procedures.