Logic-Based Delivery of Site-Specifically-Modified Proteins from Gels through Engineered Biomacromolecular Architecture

Thesis (Master's)--University of Washington, 2016-08

Porous self-protonating spiropyran-based NIPAAm gels with improved reswelling kinetics

The final publication is available at Springer via http://dx.doi.org/[10.1007/s10853-015-9458-2]

Evaluation of Protective Effect of a Water-In-Oil Microemulsion Incorporating Quercetin Against UVB-Induced Damage in Hairless Mice Skin

Purpose. Histological aspects were considered in order to evaluate the in vivo photoprotective effect of a w/o microemulsion containing quercetin against UVB irradiation-induced dermal damages. The toxicity in cell culture and the potential skin irritation resulting from topical application of this formulation were investigated. Methods. Mouse dorsal surfaces were treated topically with 300 mg of the unloaded and quercetin-loaded (0.3%, w/w) microemulsions before and after exposure to UVB (2.87 J/cm(2)) irradiation. The untreated control groups irradiated and non-irradiated were also evaluated. UVB-induced histopathological changes as well as the photoprotective effect of this formulation were evaluated considering the parameters of infiltration of inflammatory cells, epidermis thickening (basale and spinosum layers) and collagen and elastic fiber contents. The cytotoxicity of the reported formulation was evaluated in L929 mice fibroblasts by MTT assay and the skin irritation was investigated after topical application of both unloaded and quercetin-loaded microemulsions once a day for 15 days. Results. The results demonstrated that the w/o microemulsion containing quercetin reduced the incidence of histological skin alterations, mainly the connective-tissue damage, induced by exposure to UVB irradiation. This suggests that protective effects of this formulation against UV-induced responses are not secondary to the interference of UV transmission (i.e., blocking the UVB radiation from being absorbed by the skin), as is usually implied with UVB absorbers and sunscreens, but is instead due to different biological effects of this flavonoid. Furthermore, by evaluating the cytotoxic effect on L929 cells and histological aspects such as infiltration of inflammatory cells and epidermis thickness of hairless mice, the present study also demonstrated the lack of toxicity of the proposed system. Conclusion. Based on these mice models, a detailed characterization of the w/o microemulsion incorporating quercetin effects as a photochemoprotective agent on human skin is presented.

Species identification in mammals from mixed biological samples based on mitochondrial DNA control region length polymorphism.

The ability to identify the species origin of an unknown biological sample is relevant in the fields of human and wildlife forensics. However, the detection of several species mixed in the same sample still remains a challenge. We developed and tested a new approach for mammal DNA identification in mixtures of two or three species, based on the analysis of mitochondrial DNA control region interspecific length polymorphism followed by direct sequencing. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and is not based on a pre-defined panel of species. Amplicons can be separated either on agarose gels or using CE. The advantages and limitations of the assay are discussed under different conditions, such as variable template concentration, amplicon sizes and size difference among the amplicons present in the mixture. For the first time, this protocol provides a simple, reliable and flexible method for simultaneous identification of multiple mammalian species from mixtures, without any prior knowledge of the species involved.

Anells de Liesegang (experimentació amb gels)

Treball de recerca realitzat per un alumne d'ensenyament secundari i guardonat amb un Premi CIRIT per fomentar l'esperit científic del Jovent l'any 2009. Aquest treball de recerca es basa en l'experimentació i, posteriorment, l'obtenció i anàlisi de resultats de l'experiment creador d'anells de Liesegang. Aquest experiment, consistent en la precipitació d'un compost en una base gelificada formant anells distanciats logarítmicament els uns dels altres, ha estat durant més d'un segle objecte d'investigació de moltíssims científics, els quals no han sabut mai treure'n una explicació lògica i raonable d'aquest rar comportament. L'autor ha pretès recrear els curiosos anells intentant formar-los amb diferents inhibidors i compostos als trobats en la bibliografia. Després de realitzar més d'una trentena d'experiments, s'ha realitzat una anàlisi exhaustiva dels resultats. Aquest apartat ha estat un dels més enriquidors, ja que s'han dut a terme en ell comparacions sorprenents i troballes molt curioses, com per exemple la similitud entre els anells de Liesegang i les estructures de Turing, la qual intenta explicar les formes presents en els ocels dels éssers vius; i l'aparició d'anells de Liesegang segons l’òptica visual, efecte inexistent en l’àmplia bibliografia consultada. A més a més, també s'han efectuat una sèrie d'estudis: un en què es confirmen les distàncies logarítmiques entre els anells i on es realitza una comparació entre les dades empíriques i el patró matemàtic; i un altre en què s'estudia el comportament dels anells al variar els factors que regulen la velocitat de reacció.

A NOVEL POLYACRYLAMIDE-BASED HYDROGEL CROSSLINKED WITH CELLULOSE ACETATE AND PREPARED BY PRECIPITATION POLYMERIZATION

The synthesis of polyacrylamide-cellulose acetate hydrogels by precipitation polymerization in acetone solution is reported herein. These hydrogels exhibit smaller swelling ratios and larger compression moduli than homo polyacrylamide hydrogels. For cellulose acetate concentrations above 20 wt.%, hydrogels with N,N'-methylenebisacrylamide as a crosslinker exhibit swelling ratios and compression moduli similar to those of the hydrogels without the crosslinker. A possible explanation for this behavior is that cellulose acetate crosslinks polyacrylamide via free-radical reaction. The hydrogels obtained without the N,N'-methylenebisacrylamide crosslinker exhibit compression moduli up to 1.7 MPa, making them suitable for tissue engineering applications such as cartilage replacement.

Aqueous gels of mixtures of ionic surfactant SDS with pluronic copolymers P123 or F127

Gel diagrams based on tube inversion and oscillatory rheometry are reported for Pluronic copolymers F127 (E98P67E98) and P123 (E21P67E21) in mixtures with anionic surfactant sodium dodecyl sulfate (SDS). Total concentrations (e, SDS+copolymer) were as high as 50 wt% with mole ratios SDS/copolymer (mr) in the ranges 1-5 (F127) a lid 1-7 (PI 23). Temperatures were its high as 90 degrees C. Determination of the temperature dependences of the dynamic moduli served to confirm the gel boundaries from tube inversion and to reveal the high elastic moduli of the gels, e.g., compared at corn parable positions in the gel phase, a 50 wt% SDS/P123 wit h mr = 7 had G' three times that of a corresponding gel of P123 alone. Sin all-angle X-ray scattering (SAX S) was used to show that the structures of all the SDS/F127 gels were bee and that the structures of the SDS/P123 gels with mr = I were either fcc(c = 30 wt%) or hex (c = 40 wt%). Assignment of structures to SDS/P123 gels with values of mr in the range 3-7 was more difficult, as high-order scattering peaks Could be very weak, and at the higher values of c and mr, the SAXS peaks included multiple reflections.

Self-assembly of two-component gels: stoichiometric control and component selection

Two-component systems capable of self-assembling into soft gel-phase materials are of considerable interest due to their tunability and versatility. This paper investigates two-component gels based on a combination of a L-lysine-based dendron and a rigid diamine spacer (1,4-diaminobenzene or 1,4-diaminocyclohexane). The networked gelator was investigated using thermal measurements, circular dichroism, NMR spectroscopy and small angle neutron scattering (SANS) giving insight into the macroscopic properties, nanostructure and molecular-scale organisation. Surprisingly, all of these techniques confirmed that irrespective of the molar ratio of the components employed, the "solid-like" gel network always consisted of a 1:1 mixture of dendron/diamine. Additionally, the gel network was able to tolerate a significant excess of diamine in the "liquid-like" phase before being disrupted. In the light of this observation, we investigated the ability of the gel network structure to evolve from mixtures of different aromatic diamines present in excess. We found that these two-component gels assembled in a component-selective manner, with the dendron preferentially recognising 1,4-diaminobenzene (>70%). when similar competitor diamines (1,2- and 1,3-diaminobenzene) are present. Furthermore, NMR relaxation measurements demonstrated that the gel based oil 1,4-diaminobenzene was better able to form a selective ternary complex with pyrene than the gel based oil 1,4-diaminocyclohexane, indicative of controlled and selective pi-pi interactions within a three-component assembly. As such, the results ill this paper demonstrate how component selection processes in two-component gel systems call control hierarchical self-assembly.

Phospholipid-based microemulsions suitable for use in foods

The preparation of nonaqueous microemulsions using food-acceptable components is reported. The effect of oil on the formation of microemulsions stabilized by lecithin (Epikuron 200) and containing propylene glycol as immiscible solvent was investigated. When the triglycerides were used as oil, three types of phase behavior were noted, namely, a two-phase cloudy region (occurring at low lecithin concentrations), a liquid crystalline (LC) phase (occurring at high surfactant and low oil concentrations), and a clear monophasic microemulsion region. The extent of this clear one-phase region was found to be dependent upon the molecular volume of the oil being solubilized. Large molecular volume oils, such as soybean and sunflower oils, produced a small microemulsion region, whereas the smallest molecular volume triglyceride, tributyrin, produced a large, clear monophasic region. Use of the ethyl ester, ethyl oleate, as oil produced a clear, monophasic region of a size comparable to that seen with tributyrin. Substitution of some of the propylene glycol with water greatly reduced the extent of the clear one-phase region and increased the extent of the liquid crystalline region. In contrast, ethanol enhanced the clear, monophasic region by decreasing the LC phase. Replacement of some of the lecithin with the micelle-forming nonionic surfactant Tween 80 to produce mixed lecithin/Tween 80 mixtures of weight ratios (Km) 1:2 and 1:3 did not significantly alter the phase behavior, although there was a marginal increase in the area of the two-phase, cloudy region of the phase diagram. The use of the lower phosphatidylcholine content lecithin, Epikuron 170, in place of Epikuron 200 resulted in a reduction in the LC region for all of the systems investigated. In conclusion, these studies show that it is possible to prepare one-phase, clear lecithin-based microemulsions over a wide range of compositions using components that are food-acceptable.

Time-dependent gel to gel transformation of a peptide based supramolecular gelator

A dipeptide with a long fatty acid chain at its N-terminus gives hydrogels in phosphate buffer in the pH range 7.0–8.5. The hydrogel with a gelator concentration of 0.45% (w/v) at pH 7.46 (physiological pH) provides a very good platform to study dynamic changes within a supramolecular framework as it exhibits remarkable change in its appearance with time. Interestingly, the first formed transparent hydrogel gradually transforms into a turbid gel within 2 days. These two forms of the hydrogel have been thoroughly investigated by using small angle X-ray scattering (SAXS), powder X-ray diffraction (PXRD), field emission scanning electron microscopic (FE-SEM) and high-resolution transmission electron microscopic (HR-TEM) imaging, FT-IR and rheometric analyses. The SAXS and low angle PXRD studies substantiate different packing arrangements for the gelator molecules for these two different gel states (the freshly prepared and the aged hydrogel). Moreover, rheological studies of these two gels reveal that the aged gel is stiffer than the freshly prepared gel.

In situ gelling systems based on Pluronic F127/Pluronic F68 formulations for ocular drug delivery

This study evaluated the use of Pluronic F127 and Pluronic F68 as excipients for formulating in situ gelling systems for ocular drug delivery. Thermal transitions have been studied in aqueous solutions of Pluronic F127, Pluronic F68 as well as their binary mixtures using differential scanning calorimetry, rheological measurements, and dynamic light scattering. It was established that the formation of transparent gels at physiologically relevant temperatures is observed only in the case of 20 wt % of Pluronic F127. The addition of Pluronic F68 to Pluronic F127 solutions increases the gelation temperature of binary formulation to above physiological range of temperatures. The biocompatibility evaluation of these formulations using slug mucosa irritation assay and bovine corneal erosion studies revealed that these polymers and their combinations do not cause significant irritation. In vitro drug retention study on glass surfaces and freshly excised bovine cornea showed superior performance of 20 wt % Pluronic F127 compared to other formulations. In addition, in vivo studies in rabbits demonstrated better retention performance of 20 wt % Pluronic F127 compared to Pluronic F68. These results confirmed that 20 wt % Pluronic F127 offers an attractive ocular formulation that can form a transparent gel in situ under physiological conditions with minimal irritation.

Film forming solutions based on gelatin and poly(vinyl alcohol) blends: Thermal and rheological characterizations

The objective of this work was to study the theological and thermal properties of film forming solutions (FFS) based on blends of gelatin and poly(vinyl alcohol) (PVA). The effect of the PVA concentration and plasticizer presence on the flow behavior, and viscoelastic and thermal properties of FFS was studied by steady-shear flow and oscillatory experiments, and also, by microcalorimetry. The FB presented Newtonian behavior at 30 degrees C, and the viscosity was not affected neither by the PVA concentration nor by the plasticizer. All FFS presented a phase transition during tests applying temperature scanning. It was verified that the PVA affected the viscoelastic properties of FFS by dilution of gelatin. This behavior was confirmed by microcalorimetric analysis. The behaviors of the storage (G`) and loss (G ``) moduli as a function of frequency of FFS obtained at 5 degrees C were typical of physical gels; with the G` higher than the G ``. The strength of the gels was affected by the PVA concentration. (C) 2009 Elsevier Ltd. All rights reserved.

Mechanosensitivity of astrocytes on optimized polyacrylamide gels analyzed by quantitative morphometry

Cells are able to detect and respond to mechanical cues from their environment. Previous studies have investigated this mechanosensitivity on various cell types, including neural cells such as astrocytes. In this study, we have carefully optimized polyacrylamide gels, commonly used as compliant growth substrates, considering their homogeneity in surface topography, mechanical properties, and coating density, and identified several potential pitfalls for the purpose of mechanosensitivity studies. The resulting astrocyte response to growth on substrates with shear storage moduli of G` = 100 Pa and G` = 10 kPa was then evaluated as a function of coating density of poly-D-lysine using quantitative morphometric analysis. Astrocytes cultured on stiff substrates showed significantly increased perimeter, area, diameter, elongation, number of extremities and overall complexity if compared to those cultured on compliant substrates. A statistically significant difference in the overall morphological score was confirmed with an artificial intelligence-based shape analysis. The dependence of the cells` morphology on PDL coating density seemed to be weak compared to the effect of the substrate stiffness and was slightly biphasic, with a maximum at 10-100 mu g ml(-1) PDL concentration. Our finding suggests that the compliance of the surrounding tissue in vivo may influence astrocyte morphology and behavior.

Fluorescent AlPO(4) gels and glasses doped with Rhodamine 6G: Preparation, structural and optical characterization

Fluorescent AlPO(4) xerogels doped with different amounts of Rhodamine 6G (Rh6G) laser dye were prepared by a one-step sal-gel process. In addition, mesoporous AlPO(4) glasses obtained from undoped gels were loaded with different amounts of Rh6G by wet impregnation. Optical excitation and emission spectra of both series of samples show significant dependences on Rh6G concentration, revealing the influence of dye molecular aggregation. At comparable dye concentrations the aggregation effects are found to be significantly stronger in the gels than in the mesoporous glasses. This effect might be attributed to stronger interactions between the dye molecules and the glass matrix, resulting in more efficient dye dispersion in the latter. The interaction of Rh6G with the glassy AlPO(4) network has been probed by (27)Al and (31)P solid-state NMR techniques. New five- and six-coordinated aluminum environments have been observed and characterized by advanced solid-state NMR techniques probing (27)Al-(1)H and (27)Al-(31)P internuclear dipole couplings. The fractional area of these new Al sites is correlated with the combined fractional area of two new Q(3Al)((0)) and Q(2Al)((0)) phosphate species observed in the (31)P MAS NMR spectra. Based on this correlation as well as detailed composition dependent studies, we suggest that the new signals arise from the breakage of Al-O-P linkages associated with the insertion process. (C) 2010 Elsevier B.V. All rights reserved.

Structural matching of 2D electrophoresis gels using deformed graphs

2D electrophoresis is a well-known method for protein separation which is extremely useful in the field of proteomics. Each spot in the image represents a protein accumulation and the goal is to perform a differential analysis between pairs of images to study changes in protein content. It is thus necessary to register two images by finding spot correspondences. Although it may seem a simple task, generally, the manual processing of this kind of images is very cumbersome, especially when strong variations between corresponding sets of spots are expected (e.g. strong non-linear deformations and outliers). In order to solve this problem, this paper proposes a new quadratic assignment formulation together with a correspondence estimation algorithm based on graph matching which takes into account the structural information between the detected spots. Each image is represented by a graph and the task is to find a maximum common subgraph. Successful experimental results using real data are presented, including an extensive comparative performance evaluation with ground-truth data. (C) 2010 Elsevier B.V. All rights reserved.