Functional and safe encapsulation of Escherichia coli in Pluronic hydrogels for engineered living materials

Bacterial growth and metabolic activity are sensitive to the mechanical properties of their environment. Understanding how the 3D spatial confinement regulates the cell behavior is crucial not only for understanding biofilm development but also for the design and safe application of engineered materials containing living cells. This Thesis explores the use of Pluronic-based hydrogels to encapsulate genetically modified Escherichia coli bacteria. Hydrogels with different viscoelastic properties were prepared by mixing Pluronic and Pluronic diacrylate components in different ratios, giving physical hydrogels with variable degree of covalent crosslinking and different mechanical responses. Rheological properties of the hydrogels as well as the growth rate and morphology of the embedded bacterial colonies were characterized. The results provided correlations between material parameters and bacterial cell responses. Further, a bilayer thin film model was developed for long term encapsulation of the organisms, preventing leakage of cells for up to two weeks while maintaining their activity as drug/protein eluting devices or biosensing units. The bacterial bilayer thin films did not elicit significant immune responses in primary immune cells from healthy donors. The results of this Thesis demonstrate the potential of Pluronic-based biohybrid as a suitable and safe prototype for further in vitro and in vivo testing of engineered living material designs.Wachstum und Stoffwechselaktivität von Bakterien sind sensitiv gegenüber den mechanischen Eigenschaften ihrer Umgebung. Das Verständnis, wie der räumliche 3D-Einschluss das Zellverhalten reguliert, ist sowohl für die Entwicklung von Biofilmen als auch für das Design und die sichere Anwendung von technischen Materialien, die lebende Zellen enthalten, essenziell. Diese Thesis untersucht die Verwendung von Hydrogelen auf Pluronic-Basis zur Verkapselung von genetisch veränderten Escherichia coli Bakterien. Durch die Mischung von Pluronic und Pluronic-Diacrylat in verschiedenen Verhältnissen wurden physische Hydrogele mit unterschiedlichem kovalenten Vernetzungsgrad und viskoelastischen Eigenschaften hergestellt. Die Charakterisierung der rheologischen Eigenschaften der Hydrogele sowie der Wachstumsrate und Morphologie der eingebetteten Bakterien zeigte eine Korrelation zwischen den Materialparametern und dem Zellverhalten. Darüber hinaus wurde ein Doppelschicht-Dünnfilmmodell entwickelt, in dem die Organismen bis zu zwei Wochen ohne Austreten eingeschlossen wurden, während gleichzeitig Medikamenten-, Proteinfreisetzung oder die Aktivität der Zellen als Biosensoren beibehalten wird. Das Modell löste bei primären Immunzellen von gesunden Spendern keine signifikanten Immunreaktionen aus. Diese Thesis zeigt das Potenzial von Biohybriden auf Pluronic-Basis als geeigneten und sicheren Prototyp für weitere in vitro und in vivo Tests von technischen lebenden Materialien

Regulating bacterial behavior within hydrogels of tunable viscoelasticity

Engineered living materials (ELMs) are a new class of materials in which living organism incorporated into diffusive matrices uptake a fundamental role in material’s composition and function. Understanding how the spatial confinement in 3D affects the behavior of the embedded cells is crucial to design and predict ELM’s function, regulate and minimize their environmental impact and facilitate their translation into applied materials. This study investigates the growth and metabolic activity of bacteria within an associative hydrogel network (Pluronic-based) with mechanical properties that can be tuned by introducing a variable degree of acrylate crosslinks. Individual bacteria distributed in the hydrogel matrix at low density form functional colonies whose size is controlled by the extent of permanent crosslinks. With increasing stiffness and decreasing plasticity of the matrix, a decrease in colony volumes and an increase in their sphericity is observed. Protein production surprisingly follows a different pattern with higher production yields occurring in networks with intermediate permanent crosslinking degrees. These results demonstrate that, bacterial mechanosensitivity can be used to control and regulate the composition and function of ELMs by thoughtful design of the encapsulating matrix, and by following design criteria with interesting similarities to those developed for 3D culture of mammalian cells

Bio-analytical method validation and its importance in pharma research - A review article

Bioanalytical method based on a variety of physico-chemical and biological techniques such as chromatography, immunoassay and mass spectrometry, must be validated prior to and during use to give confidence in the results generated. It is the process used to establish that a quantitative analytical method is suitable for biomedical applications. Bioanalytical method validation includes all of the procedures that demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum, or urine is reliable and reproducible for the intended use. The present manuscript focuses on the consistent evaluation of the key bioanalytical validation parameters is discussed: accuracy, precision, sensitivity, selectivity, standard curve, limits of quantification, range, recovery and stability. These validation parameters are described, together with an example of validation methodology applied in the case of chromatographic methods used in bioanalysis, taking in account to the recent Food and Drug Administration (FDA) guidelines and EMA guid

Validated RP-HPLC Method for Simultaneous Quantitation of Losartan Potassium and Metolazone in Bulk Drug and Formulation

A HPLC method has been described for simultaneous determination of Losartan potassium and Metolazone in formulation. This method is based on a HPLC separation of the two drugs on the Thermo Hypersil BDS–C18 (250 mm × 4.6 mm, 5.0 μm) with isocratic conditions and a simple mobile phase containing acetonitrile:water (60:40) at a flow rate of 0.8 mL/min using UV detection at 237 nm. This method has been applied to a marketed formulation without interference of excipients. The linear regression analysis data for the calibration plots showed a good linear relationship over the concentration range of 2–12 μg/mL for Losartan potassium and 0.2–1.2 μg/mL for Metolazone, respectively. The method was validated for precision, robustness and recovery. Statistical analysis showed that the method is repeatable and selective for the estimation of Losartan potassium and Metolazone

Formulation and Evaluation of Gastro Retentive Mucoadhesive Sustained Release Pellets of Acyclovir

Acyclovir is an antiviral drug, belonging to the deoxyguanosine family, widely prescribed for the treatment of herpes simplex viral infections, as well as in the treatment of herpes zoster (shingles). Oral bioavailability of acyclovir is very low (10–20%) owing to its first pass metabolism with elimination half-life (t1/2) of 2-3 h. It has absorption window in upper gastrointestinal tract. Due to its rapid elimination from site of absorption and short biological half life, sustained release formulation system for acyclovir is advantageous. In this study, gastro retentive muco-adhesive SR pellets of acyclovir was prepared using HPMC K 100M as matrix former and Sodium CMC as mucoadhesive polymer by extrusion spheronization technique. Acyclovir pellets prepared with higher concentration of HPMC (batch G) showed in vitro drug release for 12 h with sufficient mucoadhesion strength and ex vivo resident time. Release kinetic studies indicated that drug release data had best fit to Higuchi’s model. In-vivo studies in rat model proved that relative bioavailability of acyclovir SR pellets get increased by 1.98 fold as compared plain drug suspension. The optimized formulation batch G was found to be stable during six months accelerated stability period

Effects of fescue toxicosis and chronic heat stress on murine hepatic gene expression

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Title from title screen of research.pdf file (viewed on July 31, 2007)Vita.Includes bibliographical references.Thesis (Ph. D.) University of Missouri-Columbia 2006.Dissertations, Academic -- University of Missouri--Columbia -- Animal sciences.Fescue toxicosis affects domestic animals grazing fescue pasture infected with the endophytic fungus, Neotyphodium coenophialum. Signs of fescue toxicosis include increased body temperature and respiration rate and decreased milk yield and reproductive performance. Laboratory mice also exhibit symptoms of fescue toxicosis as indicated by reduced growth rate and reproductive performance. Mice were used to study effects of fescue toxicosis on hepatic gene expression. Twenty-seven mice were randomly allocated to a diet containing either 50% endophyte-infected (E+) or endophyte-free (E-) fescue seed for two wks under thermoneutral conditions. A two-stage ANOVA of microarray data identified thirty-six genes differentially expressed between mice fed E+ and E- diets. The E+ diet resulted in down-regulation of genes involved in sex-steroid pathway and in cholesterol and lipid metabolism. Genes coding for ribosomes and protein synthesis were up-regulated by the E+ diet. Mice were also used to study the effects of chronic heat stress on hepatic gene expression. Twenty-five mice were randomly allocated to either chronic heat stress (cHS; 34 ± 1C̕) or thermoneutral (TN; 24 ± 1C̕) conditions for a period of two wks from 47 to 60 d of age. A two-stage ANOVA of 1353 gene oligoarray data identified thirty genes as differentially expressed due to cHS. Genes involved in the anti-oxidant pathway were up-regulated due to cHS. Genes involved in generation of reactive oxygen radicals and a number of mitochondrial expressed genes were down-regulated by cHS. However, cHS did not produce an increase in oxidative stress induced mitochondrial DNA damage. Furthermore, effects of heat stress on changes in gene expression due to fescue toxicosis in mice liver were studied using DNA microarrays. Our goal was to characterize the differences in liver gene expression of mice exposed to chronic heat stress (cHS) and E+ when compared to mice fed E+ at TN. Mice were fed E+ diet under cHS (34 ± 1C̕; n = 13; E+cHS) or TN conditions (24 ± 1C̕; n = 14; E+TN) for a period of two wks between 47 to 60 d of age. Forty-one genes were differentially expressed between treatment groups. Genes coding for phase I detoxification and anti-oxidant pathway were up-regulated in E+cHS mouse liver. Key genes involved in de novo lipogenesis and lipid transport were also up-regulated. Finally, genes involved in DNA damage control and unfolded protein responses were down-regulated. In summary, mice fed an E+ diet at TN resulted in change in expression of genes involved in sex-steroid pathway while this pathway was not perturbed in mice exposed to cHS or to E+cHS treatments. Changes in expression of genes involved in lipid and cholesterol metabolism pathway occurred in mice exposed to E+ and to E+cHS treatment. Anti-oxidant gene expression changes occurred in mice exposed to cHS and to E+cHS, but not in E+ treated mice. Interestingly, gene expression changes involved in the detoxification pathway were seen only in mice exposed to combination of E+ and cHS. Biological pathways and gene expression changes identified in mouse liver due to E+, cHS, and E+cHS will help to understand molecular mechanisms by which fescue toxicosis and heat stress affects animals

Optimization of process variables for phyllanthin extraction from Phyllanthus amarus leaves by supercritical fluid using a Box-Behnken experimental design followed by HPLC identification

The response surface methodology using the Box-Behnken design was established to describe supercritical carbon dioxide assisted extraction of phyllanthin from Phyllanthus amarus Schum and Thonn leaves prior to HPLC analysis. The effects of extraction pressure, temperature, modifier concentration and extraction time on the yield of phyllanthin were investigated. By solving the regression equation, the optimum conditions were as follows: extraction pressure 23.2 MPa, temperature 40 °C, methanol as modifier at a concentration 10 % and time 90 min. Under these conditions, the phyllanthin yield was 12.83 ± 0.28 mg g-1, which was in good agreement with the predicted values. Modifier concentration and extraction time showed a significant effect on the phyllanthin yield

Bioprospecting of Adhatoda vasica for Identification of Novel Compounds using Chromatographic Methods and Screening for Anti-diabetic and Antioxidant Activity

This investigation column eluted fractions of leaf Adhatoda vasica of was assessed for its phytochemical screening, column chromatography, thin layer chromatographic studies, protease activity, anti-inflammatory activity, antidiabetic activity and antioxidant activity. Phytochemical screening reflects the presence of alkaloid, flavonoids, coumarins, terpenoids, steroids, emodin’s, Quinone’s. Column chromatography method was used for purification of bioactive compounds. Thin layer chromatographic study was carried out by using various solvent system of different type of polarity of n- butanol, acetic acid and acetone. TLC profiling shows pure band at 254nm and 366 nm. The strong “proteolytic activity” also pointed out in purified fraction of eluted fraction. In vitro anti-inflammatory activity was evaluated using albumin denaturation fraction 3, showing highest activity 75% followed by fraction 5 (62.73%), membrane stabilization assay fraction 6 (80.23%) followed by fraction 3 (64.65%) and proteinase inhibitory activity of fraction 5(88%) followed by fraction 7 (87.68%) at concentration 500 µg/ml. Aspirin (90.87%) was used as standard drug for the study of anti-inflammatory activity. In vitro antidiabetic activity was performed using Alfa amylase inhibition assay. Highest activity was showed in fraction 4 (79.05 %) and fraction 5 (77.05 %) at concentration 500 µg/ml. Antioxidant activity was performed by reducing power assay fraction number 2 has higher absorbance 1.04 at 500µg/ml followed by reducing power of column eluted fraction was compared with ascorbic acid as standard showing higher absorbance 0.93 at 500µg/ml

Conceptual Study Of Goghrita eye drops (Aschyotana)in Computer Vision Syndrome

Conceptual study of Goghrita Eye drops (Aschyotana) in Computer Vision Syndrome.                     Dr .Santosh .S. Mulik* , Dr. Dilip P.Bhusari  KEYWORDSComputer Vision Syndrome ,Goghrita, Aschyotana, Eye dropsABSTRACTAccording to the National Institute of occupational safety and health ,Computer vision syndrome ( C V S ) affects 90% of the people who spend 3 hours or more a day at computer .With the advent of computer , users confront new challenges both at their work place and school systems. Computer is a heat factory . By working for long hours using a computer monitor, a complication of vision and ophthalmic symptoms may develop. They are collectively known as Computer Vision Syndrome. Lubricating drops can reduce the effects of dry eye in computer vision syndrome. But its preservatives are harmful to eye. So long term use is not possible and effects of lubricating drops are temporary. A recent study in Japan revealed that the majority of lubricating eye drop users were dissatisfied with the therapeutic effects. C V S syndrome includes burning sensation ,dryness, redness  and itching in eyes..These symptoms related to Netradaha, Netrarukshtava, Netralalima, Netrakandu in Ayurveda.Goghrita is Snehonattam. It is Rasayana And Chakshushya .It has properties of Snigdha, Guru and Mrudu.Because of these properties Goghrita is very useful for vitiated pitta and vatta dosha in CVS . It has lubricating properties which may be useful in reducing the symptoms of  computer vision syndrome. It contains vitamin A 3500/100gm, beta-carotene and Vit E. Vitamin A keeps the outer lining of eye ball moist and prevent blindness. Beta-carotene and Vit E which are well known antioxidants.         So Goghrita Eye drops(Aschyotana )can be used as alternative treatment in CVS.

Printed Degradable Optical Waveguides for Guiding Light into Tissue

Optogenetics and photonic technologies are changing the future of medicine. To implement light‐based therapies in the clinic, patient‐friendly devices that can deliver light inside the body while offering tunable properties and compatibility with soft tissues are needed. Here extrusion printing of degradable, hydrogel‐based optical waveguides with optical losses as low as 0.1 dB cm−1 at visible wavelengths is described. Core‐only and core‐cladding fibers are printed at room temperature from polyethylene glycol (PEG)‐based and PEG/Pluronic precursors, and cured by in situ photopolymerization. The obtained waveguides are flexible, with mechanical properties tunable within a tissue‐compatible range. Degradation times are also tunable by adjusting the molar mass of the diacrylate gel precursors, which are synthesized by linking PEG diacrylate (PEGDA) with varying proportions of DL‐dithiothreitol (DTT). The printed waveguides are used to activate photochemical and optogenetic processes in close‐to‐physiological environments. Light‐triggered migration of cells in a photoresponsive 3D hydrogel and drug release from an optogenetically‐engineered living material by delivering light across >5 cm of muscle tissue are demonstrated. These results quantify the in vitro performance, and reflect the potential of the printed degradable fibers for in vivo and clinical applications