Slow light optofluidics:a proposal

The resonant slow light structures created along a thin-walled optical capillary by nanoscale deformation of its surface can perform comprehensive simultaneous detection and manipulation of microfluidic components. This concept is illustrated with a model of a 0.5 mm long, 5 nm high, triangular bottle resonator created at a 50 μm radius silica capillary containing floating microparticles. The developed theory shows that the microparticle positions can be determined from the bottle resonator spectrum. In addition, the microparticles can be driven and simultaneously positioned at predetermined locations by the localized electromagnetic field created by the optimized superposition of eigenstates of this resonator, thus exhibiting a multicomponent, near-field optical tweezer.

Nanophotonic microfluidic sensor and manipulator

Resonant structures created along a thin capillary by nanoscale deformation of its surface can perform comprehensive sensing and manipulation of microfluids. The concept is illustrated with a model of triangular bottle resonator and floating microparticles.

Innovative biomarkers for predicting type 2 diabetes mellitus:relevance to dietary management of frailty in older adults

Type 2 diabetes mellitus (T2DM) increases in prevalence in the elderly. There is evidence for significant muscle loss and accelerated cognitive impairment in older adults with T2DM; these comorbidities are critical features of frailty. In the early stages of T2DM, insulin sensitivity can be improved by a “healthy” diet. Management of insulin resistance by diet in people over 65 years of age should be carefully re-evaluated because of the risk for falling due to hypoglycaemia. To date, an optimal dietary programme for older adults with insulin resistance and T2DM has not been described. The use of biomarkers to identify those at risk for T2DM will enable clinicians to offer early dietary advice that will delay onset of disease and of frailty. Here we have used an in silico literature search for putative novel biomarkers of T2DM risk and frailty. We suggest that plasma bilirubin, plasma, urinary DPP4-positive microparticles and plasma pigment epithelium-derived factor merit further investigation as predictive biomarkers for T2DM and frailty risk in older adults. Bilirubin is screened routinely in clinical practice. Measurement of specific microparticle frequency in urine is less invasive than a blood sample so is a good choice for biomonitoring. Future studies should investigate whether early dietary changes, such as increased intake of whey protein and micronutrients that improve muscle function and insulin sensitivity, affect biomarkers and can reduce the longer term complication of frailty in people at risk for T2DM.

The relationship of systemic markers of renal function and vascular function with retinal blood vessel responses

Purpose: To test the hypothesis of a significant relationship between systemic markers of renal and vascular function (processes linked to cardiovascular disease and its development) and retinal microvascular function in diabetes and/or cardiovascular disease.Methods: Ocular microcirculatory function was measured in 116 patients with diabetes and/or cardiovascular disease using static and continuous retinal vessel responses to three cycles of flickering light. Endothelial function was evaluated by von Willebrand factor (vWf), endothelial microparticles and soluble E selectin, renal function by serum creatinine, creatinine clearance and estimated glomerular filtration rate (eGFR). HbA1c was used as a control index.Results: Central retinal vein equivalence and venous maximum dilation to flicker were linked to HbA1c (both p<0.05). Arterial reaction time was linked to serum creatinine (p=0.036) and eGFR (p=0.039), venous reaction time was linked to creatinine clearance (p=0.018). Creatinine clearance and eGFR were linked to arterial maximum dilatation (p<0.001 and p=0.003 respectively) and the dilatation amplitude (p=0.038 and p=0.048 respectively) responses in the third flicker cycle. Of venous responses to the first flicker cycle, HbA1c was linked to the maximum dilation response (p=0.004) and dilatation amplitude (p=0.017), vWf was linked to the maximum constriction response (p=0.016), and creatinine clearance to the baseline diameter fluctuation (p=0.029). In the second flicker cycle, dilatation amplitude was linked to serum creatinine (p=0.022). Conclusions: Several retinal blood vessel responses to flickering light are linked to glycaemia and renal function, but only one index is linked to endothelial function. Renal function must be considered when interpreting retinal vessel responses.

Stabilization of troponin I for applications in clinical chemistry and forensic medicine. Cardiac troponin I: A time of death marker

Cardiac troponin I (cTnI) is one of the most useful serum marker test for the determination of myocardial infarction (MI). The first commercial assay of cTnI was released for medical use in the United States and Europe in 1995. It is useful in determining if the source of chest pains, whose etiology may be unknown, is cardiac related. Cardiac TnI is released into the bloodstream following myocardial necrosis (cardiac cell death) as a result of an infarct (heart attack). In this research project the utility of cardiac troponin I as a potential marker for the determination of time of death is investigated. The approach of this research is not to investigate cTnI degradation in serum/plasma, but to investigate the proteolytic breakdown of this protein in heart tissue postmortem. If our hypothesis is correct, cTnI might show a distinctive temporal degradation profile after death. This temporal profile may have potential as a time of death marker in forensic medicine. The field of time of death markers has lagged behind the great advances in technology since the late 1850's. Today medical examiners are using rudimentary time of death markers that offer limited reliability in the medico-legal arena. Cardiac TnI must be stabilized in order to avoid further degradation by proteases in the extraction process. Chemically derivatized magnetic microparticles were covalently linked to anti-cTnI monoclonal antibodies. A charge capture approach was also used to eliminate the antibody from the magnetic microparticles given the negative charge on the microparticles. The magnetic microparticles were used to extract cTnI from heart tissue homogenate for further bio-analysis. Cardiac TnI was eluted from the beads with a buffer and analyzed. This technique exploits banding pattern on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a western blot transfer to polyvinylidene fluoride (PVDF) paper for probing with anti-cTnI monoclonal antibodies. Bovine hearts were used as a model to establish the relationship of time of death and concentration/band-pattern given its homology to human cardiac TnI. The final concept feasibility was tested with human heart samples from cadavers with known time of death. ^

The non-linear acousto-mechanical and thermodynamic response of spherical absorbers to laser pulses of various temporal profiles

This study is to theoretically investigate shockwave and microbubble formation due to laser absorption by microparticles and nanoparticles. The initial motivation for this research was to understand the underlying physical mechanisms responsible for laser damage to the retina, as well as the predict threshold levels for damage for laser pulses with of progressively shorter durations. The strongest absorbers in the retina are micron size melanosomes, and their absorption of laser light causes them to accrue very high energy density. I theoretically investigate how this absorbed energy is transferred to the surrounding medium. For a wide range of conditions I calculate shockwave generation and bubble growth as a function of the three parameters; fluence, pulse duration and pulse shape. In order to develop a rigorous physical treatment, the governing equations for the behavior of an absorber and for the surrounding medium are derived. Shockwave theory is investigated and the conclusion is that a shock pressure explanation is likely to be the underlying physical cause of retinal damage at threshold fluences for sub-nanosecond pulses. The same effects are also expected for non-biological micro and nano absorbers. ^

Improved estimation of postmortem interval with Cardiac Troponin T and improved analytical methods

Accurate knowledge of the time since death, or postmortem interval (PMI), has enormous legal, criminological, and psychological impact. In this study, an investigation was made to determine whether the relationship between the degradation of the human cardiac structure protein Cardiac Troponin T and PMI could be used as an indicator of time since death, thus providing a rapid, high resolution, sensitive, and automated methodology for the determination of PMI. ^ The use of Cardiac Troponin T (cTnT), a protein found in heart tissue, as a selective marker for cardiac muscle damage has shown great promise in the determination of PMI. An optimized conventional immunoassay method was developed to quantify intact and fragmented cTnT. A small sample of cardiac tissue, which is less affected than other tissues by external factors, was taken, homogenized, extracted with magnetic microparticles, separated by SDS-PAGE, and visualized with Western blot by probing with monoclonal antibody against cTnT. This step was followed by labeling and available scanners. This conventional immunoassay provides a proper detection and quantitation of cTnT protein in cardiac tissue as a complex matrix; however, this method does not provide the analyst with immediate results. Therefore, a competitive separation method using capillary electrophoresis with laser-induced fluorescence (CE-LIF) was developed to study the interaction between human cTnT protein and monoclonal anti-TroponinT antibody. ^ Analysis of the results revealed a linear relationship between the percent of degraded cTnT and the log of the PMI, indicating that intact cTnT could be detected in human heart tissue up to 10 days postmortem at room temperature and beyond two weeks at 4C. The data presented demonstrates that this technique can provide an extended time range during which PMI can be more accurately estimated as compared to currently used methods. The data demonstrates that this technique represents a major advance in time of death determination through a fast and reliable, semi-quantitative measurement of a biochemical marker from an organ protected from outside factors. ^

Microspheres for liver radiomicrospheres therapy and planning

Liver cancer accounts for nearly 10% of all cancers in the US. Intrahepatic Arterial Radiomicrosphere Therapy (RMT), also known as Selective Internal Radiation Treatment (SIRT), is one of the evolving treatment modalities. Successful patient clinical outcomes require suitable treatment planning followed by delivery of the microspheres for therapy. The production and in vitro evaluation of various polymers (PGCD, CHS and CHSg) microspheres for a RMT and RMT planning are described. Microparticles with a 30±10 µm size distribution were prepared by emulsion method. The in vitro half-life of the particles was determined in PBS buffer and porcine plasma and their potential application (treatment or treatment planning) established. Further, the fast degrading microspheres (≤ 48 hours in vitro half-life) were labeled with 68Ga and/or 99mTc as they are suitable for the imaging component of treatment planning, which is the primary emphasis of this dissertation. Labeling kinetics demonstrated that 68Ga-PGCD, 68Ga-CHSg and 68Ga-NOTA-CHSg can be labeled with more than 95% yield in 15 minutes; 99mTc-PGCD and 99mTc-CHSg can also be labeled with high yield within 15-30 minutes. In vitro stability after four hours was more than 90% in saline and PBS buffer for all of them. Experiments in reconstituted hemoglobin lysate were also performed. Two successful imaging (RMT planning) agents were found: 99mTc-CHSg and 68Ga-NOTA-CHSg. For the 99mTc-PGCD a successful perfusion image was obtained after 10 minutes, however the in vivo degradation was very fast (half-life), releasing the 99mTc from the lungs. Slow degrading CHS microparticles (> 21 days half-life) were modified with p-SCN-b-DOTA and labeled with 90 Y for production of 90Y-DOTA-CHS. Radiochemical purity was evaluated in vitro and in vivo showing more than 90% stability after 72 and 24 hours respectively. All agents were compared to their respective gold standards (99mTc-MAA for 68Ga-NOTA-CHSg and 99m Tc-CHSg; 90Y-SirTEX for 90Y-DOTA-CHS) showing superior in vivo stability. RMT and RMT planning agents (Therapy, PET and SPECT imaging) were designed and successfully evaluated in vitro and in vivo.

Micropartículas de quitosana estruturadas com aerosil®: estabilidade, adsorção, encapsulação e liberação de substâncias ativas

Biodegradable microspheres used as controlled release systems are important in pharmaceutics. Chitosan biopolymer represents an attractive biomaterial alternative because of its physicochemical and biological characteristics. Chitosan microspheres are expected to become promising carrier systems for drug and vaccine delivery, especially for non-invasive ways oral, mucosal and transdermal routes. Controlling the swelling rate and swelling capacity of the hydrogel and improving the fragile nature of microspheres under acidic conditions are the key challenges that need to be overcomed in order to enable the exploration of the full pharmaceutical potential use of these microparticles. Many studies have focused on the modification of chitosan microsphere structures with cross-linkers, various polymers blends and new organic-inorganic hybrid systems in order to obtain improved properties. In this work, microspheres made of chitosan and nanosized hydrophobic silica (Aerosil R972) were produced by a method consisting of two steps. First, a preparation of a macroscopically homogeneous chitosan-hydrophobic silica dispersion was prepared followed by spray drying. FTIR spectroscopy, X-ray powder diffraction, differential scanning calorimetry, thermal gravimetric analysis, scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (TEM) were used to characterize the microspheres. Also, the were conducted acid stability, moisture sorption capacity, release properties and biological assays. The chitosan-hydrophobic silica composite microspheres showed improved thermal degradation, lower water affinity, better acid stability and ability to retard rifampicin and propranolol hydrochloride (drug models) release under simulated physiological conditions. In vitro biocompatibility studies indicated low cytotoxicity and low capacity to activate cell production of the pro-inflammatory mediator nitric oxide. The results show here encourage further studies on the use of the new chitosan-hydrophobic silica composite microspheres as drug carrier systems via oral or nasal routes.

Micropartículas contendo nanopartículas de quitosana usando sulfato e genipina para liberação modificada da triancinolona: obtenção, caracterização e estudo em células tumorais

Chitosan is a polymer biocompatibility and biodegradability widely used in drug delivery systems. The co-crosslinking of chitosan with sodium sulfate and genipin, to form particulate systems is related of making them more resistant to acidic pH and to modulate the release kinetics for the oral route. Triamcinolone is a glucocorticoid with anti-inflammatory and immunosuppressive actions. The nanoparticles were prepared by co-crosslinking and characterized for particle size, PDI, zeta potential, crosslinking degree, encapsulation rate, morphology, infrared spectroscopy, thermal analysis, release kinetics and cells studies. The nanoparticles were prepared initially without genipin with sodium sulphate and the particles parameters were monitored in function of different ratio of drug / polymer, different concentrations of sodium sulfate and polysorbate 80 and the drip mode of crosslinkers on polymers. After optimizing conditions, the chosen system parameters without genipin included mean diameter of 312.20 ± 5.70 nm, PDI 0.342 ± 0.013 and zeta potential of 20.18 ± 2.28 mV. The genipin was introduced into the system analyzing different concentrations (0.5, 1.0 and 2.0 mM) and crosslinking times (3, 6, 12 and 24 h). Evaluating crosslinking time with genipin (0.5 mM) it was showed that varying the genipin reaction time the systems size ranged from 235.1 to 334.4 nm, the PDI from 0.321 to 0.392 and zeta potential 20.92 to 30.39 mV. The crosslinking degree that coud vary from 14 to 30 %. Nanoparticles without genipina, 6 h and 24 h crosslinking time were dried by spray-drying method. Analysis by scanning electron micrograph (SEM) revealed that the microparticles showed spherical morphology. The encapsulation rate was 75 ± 2.3 % using validated HPLC methodology. The infrared analysis showed chemical interactions between the components of the formulation. Thermal analysis showed that systems with a higher degree of crosslinking had a higher thermal stability. On release kinetics, increasing the degree of crosslinking was able to decrease the concentration and rate of release of triamcinolone. In studies with liver cancer cells (HepG2) and colon (HT-29), the microparticulate prepared with triamcinolone and 24 h of crosslinking with genipin showed a potential for antitumor activity in hepatic cell line HepG2. Therefore, a new delivery system for triamcinolone on polymeric nanoparticles of chitosan cocrosslinked with genipin and sodium sulfate was obtained with hepatic antitumor potential.

Reversibly Photo-Crosslinkable Polycarbonate-Based Polymersomes for Drug Encapsulation and Delivery

This thesis describes the preparation of polymersomes from poly(ethylene glycol)-block-polycarbonate (PEG-PC) copolymers functionalized with pendant coumarin groups. Coumarin groups undergo photo-reversible dimerization when irradiated with specific ultraviolet wavelengths, so they can be used to prepare polymers with photo-responsive properties. In this case, the pendant coumarin groups enable stabilization of the polymersome membrane through photo-crosslinking of the hydrophobic block. Initially, several novel cinnamoyl and coumarin functionalized cyclic carbonate monomers were synthesized using ester, ether, or amide linkages. While the homopolymerization of these functionalized monomers proved challenging due to their high melting points, both cinnamoyl and coumarin functionalized monomers were successfully copolymerized with trimethylene carbonate (TMC) at 100 ℃ using a catalyst-free melt polymerization process where the TMC doubled as a solvent for the higher melting point monomer. Using this system, polycarbonate copolymers with up to 33% incorporation of the functionalized monomers were prepared. In addition, an investigation of some anomalous polymerization results identified previously unreported triethylamine-based catalysts for the melt polymerization of carbonate monomers. These studies also demonstrated that the catalyst-free polymerization of TMC occurs faster and at lower temperatures than previously reported. Subsequently, the photo-crosslinking of cinnamoyl and coumarin functionalized polycarbonates was compared and coumarin was identified as the more effective crosslinking agent when using 300-400 nm UV. An investigation of the photo-reversibility of the coumarin dimerization revealed no discernible change in the properties of crosslinked networks, but rapid photo-reversion in dilute solutions. The photo-crosslinking and photo-reversion kinetics of the coumarin functionalized polycarbonates were determined to be second-order in both cases. Finally, the self-assembly of PEG-PC diblock copolymers functionalized with coumarin was examined and both reverse solvent evaporation and solvent displacement were found to induce self-assembly, with hydrophilic mass fractions (f-factors) of 12-28% resulting in the formation of solid microparticles and nanoparticles and f-factors of 33-43% resulting in the formation of polymersomes. The stabilization of these polymersome membranes through photo-initiator-free photo-crosslinking was demonstrated with the crosslinking allowing polymersomes to withstand centrifugation at 12,000 x g. In addition, the encapsulation of calcein, as a model small molecule drug, in the stabilized polymersomes was successfully demonstrated using confocal microscopy.

Vaisseau sanguin reconstruit par génie tissulaire : Développement d'une nouvelle approche pour la reconstruction de la media et interaction avec les microparticules

La media vasculaire est au coeur des processus physiopathologiques qui entraînent le développement de l’athérosclérose. L’utilisation d’une media reconstruite par génie tissulaire permet d’étudier les cellules musculaires lisses (CML) humaines dans un environnement plus physiologique que les cellules en culture monocouche. Les travaux présentés dans cette thèse sont orientés autour de la media vasculaire reconstruite par génie tissulaire comme modèle d’étude pharmacologique et prothèse vasculaire autologue. La première partie des travaux porte sur l’étude des interactions de cette tunique avec les microparticules (MP) circulantes. D’abord, nous avons montré que la présence de l’adventice modifie la réponse de la media aux MP produites in vitro à partir des lymphocytes T. Ensuite, l’étude de l’effet des MP isolées du sérum de patients en choc septique sur la media humaine a démontré que ces MP sont en mesure d’augmenter la contraction de la media par un mécanisme impliquant une diminution du NO et une augmentation de l’expression de l’ARN messager de l’interleukine-10. L’incubation de la media reconstruite avec cette cytokine anti-inflammatoire bloque l’hyporéactivité induite par les lipopolysaccharides. Le même phénomène a été reproduit in vivo, chez le rongeur. Ces résultats suggèrent que les SMP auraient un effet protecteur sur la fonction vasculaire, en potentialisant la contraction de la media. Ensuite, nous avons optimisé l’approche de reconstruction de prothèses vasculaires par auto-assemblage proposée initialement pour l’adapter au contexte particulier des CML. L’objectif principal était de permettre l’étude physiopathologique de la media à partir de toutes les lignées de CML; indépendamment de leur capacité de synthèse de matrice extracellulaire. Pour ce faire, nous avons développé un échafaudage de matrice extracellulaire produit par auto-assemblage à partir de fibroblastes humains. L’utilisation de cet échafaudage génère une media plus résistante et plus contractile que la technique initiale. Enfin, une anisotropie a été créée dans cet échafaudage pour permettre une orientation physiologique des CML. La media reconstruite devient ainsi plus résistante et plus contractile. Ces améliorations permettent de reconstruire des media à partir des cellules de plus de patients et mèneront à des études pharmacologiques plus représentatives de la population. Cet échafaudage facilitera la translation clinique de ce modèle de media reconstruite par génie tissulaire.

Use of Spirulina platensis micro and nanoparticles for the removal synthetic dyes from aqueous solutions by biosorption

In this research, micro and nanoparticles of Spirulina platensis dead biomass were obtained, characterized and employed to removal FD&C red no. 40 and acid blue 9 synthetic dyes from aqueous solutions. The effects of particle size (micro and nano) and biosorbent dosage (from 50 to 750 mg) were studied. Pseudofirst order, pseudo-second order and Elovich models were used to evaluate the biosorption kinetics. The biosorption nature was verified using energy dispersive X-ray spectroscopy (EDS). The best results for both dyes were found using 250 mg of nanoparticles, in these conditions, the biosorption capacities were 295 mg g−1 and 1450 mg g−1, and the percentages of dye removal were 15.0 and 72.5% for the FD&C red no. 40 and acid blue 9, respectively. Pseudo-first order model was the more adequate to represent the biosorption of both dyes onto microparticles, and Elovich model was more appropriate to the biosorption onto nanoparticles. The EDS results suggested that the dyes biosorption onto microparticles occurred mainly by physical interactions, and for the nanoparticles, chemisorption was dominant.

First observation on neustonic plastics in waters off NW Spain (spring 2013 and 2014)

This paper examines the presence and distribution of plastic particles in waters off the NW Spanish Atlantic coast. A pilot sampling program was initiated in 2013 to study the presence of plastic particles in surface waters. A total of 41 neuston samples were collected using a manta trawl fitted with a 333 μm mesh (21 samples in 2013 and 20 samples in 2014). Several types of plastic particles were observed in 95% of the stations. A total of 1463 plastic microparticles (<5 mm; mps) and 208 mesoparticles (>5 mm and <20 mm; MPS) were counted. Average concentrations recorded were 0.034 ± 0.032 and 0.176 ± 0.278 mps m−2 and 0.005 ± 0.005 and 0.028 ± 0.043 MPS m−2, respectively for 2013 and 2014. Results on this emerging topic are discussed as a preliminary step towards implementation of the Marine Strategy Framework Directive in the region. Harmonization of protocols for determination of plastic particles is urgently needed in order to compare results between regions and to ensure coherence in the implementation of the MSFD. This aspect is also important at a worldwide scale.

Dynamic Coculture of a Prevascularized Engineered Bone Construct

The generation of functional, vascularized tissues is a key challenge for the field of tissue engineering. Before clinical implantations of tissue engineered bone constructs can succeed, in vitro fabrication needs to address limitations in large-scale tissue development, including controlled osteogenesis and an inadequate vasculature network to prevent necrosis of large constructs. The tubular perfusion system (TPS) bioreactor is an effective culturing method to augment osteogenic differentiation and maintain viability of human mesenchymal stem cell (hMSC)-seeded scaffolds while they are developed in vitro. To further enhance this process, we developed a novel osteogenic growth factors delivery system for dynamically cultured hMSCs using microparticles encapsulated in three-dimensional alginate scaffolds. In light of this increased differentiation, we characterized the endogenous cytokine distribution throughout the TPS bioreactor. An advantageous effect in the ‘outlet’ portion of the uniaxial growth chamber was discovered due to the system’s downstream circulation and the unique modular aspect of the scaffolds. This unique trait allowed us to carefully tune the differentiation behavior of specific cell populations. We applied the knowledge gained from the growth profile of the TPS bioreactor to culture a high-volume bone composite in a 3D-printed femur mold. This resulted in a tissue engineered bone construct with a volume of 200cm3, a 20-fold increase over previously reported sizes. We demonstrated high viability of the cultured cells throughout the culture period as well as early signs of osteogenic differentiation. Taking one step closer toward a viable implant and minimize tissue necrosis after implantation, we designed a composite construct by coculturing endothelial cells (ECs) and differentiating hMSCs, encouraging prevascularization and anastomosis of the graft with the host vasculature. We discovered the necessity of cell to cell proximity between the two cell types as well as preference for the natural cell binding capabilities of hydrogels like collagen. Notably, the results suggested increased osteogenic and angiogenic potential of the encapsulated cells when dynamically cultured in the TPS bioreactor, suggesting a synergistic effect between coculture and applied shear stress. This work highlights the feasibility of fabricating a high-volume, prevascularized tissue engineered bone construct for the regeneration of a critical size defect.