Armando Lucero-Acuña

Pre-exposure to nitrite in the absence of ammonium strongly inhibits anammox.

Anaerobic ammonium oxidizing bacteria (Anammox) are known to be inhibited by their substrate, nitrite. However, the mechanism of inhibition and the physiological conditions under which nitrite impacts the performance of anammox bioreactors are still unknown. This study investigates the role of pre-exposing anammox bacteria to nitrite alone on their subsequent activity and metabolism after ammonium has been added. Batch experiments were carried out with anammox granular biofilm pre-exposed to nitrite over a range of concentrations and durations in the absence of ammonium. The effect of pre-exposure to nitrite alone compared to nitrite simultaneously fed with ammonium was evaluated by measuring the anammox activity and the accumulation of the intermediate, nitric oxide. The results show that the inhibitory effect was more dramatic when bacteria were pre-exposed to nitrite in absence of ammonium, as revealed by the lower activity and the higher accumulation of nitric oxide. The nitrite concentration causing 50% inhibition was 53 and 384 mg N L(-1) in the absence or the presence of ammonium, respectively. The nitrite inhibition was thus 7.2-fold more severe in the absence of ammonium. Biomass exposure to nitrite (25 mg N L(-1)), in absence of ammonium, led to accumulation of nitric oxide. On the other hand when the biomass was exposed to nitrite in presence of ammonium, accumulation of nitric oxide was only observed at much higher nitrite concentrations (500 mg N L(-1)). The inhibitory effect of nitrite in the absence of ammonium was very rapid. The rate of decay of the anammox activity was equivalent to the diffusion rate of nitrite up to 46% of activity loss. The results taken as a whole suggest that nitrite inhibition is more acute when anammox cells are not actively metabolizing. Accumulation of nitric oxide in the headspace most likely indicates disruption of the anammox biochemistry by nitrite inhibition, caused by an interruption of the hydrazine synthesis step.

Nanoparticle delivery of an AKT/PDK1 inhibitor improves the therapeutic effect in pancreatic cancer

The K-ras mutation in pancreatic cancer can inhibit drug delivery and increase drug resistance. This is exemplified by the therapeutic effect of PH-427, a small molecule inhibitor of AKT/PDK1, which has shown a good therapeutic effect against a BxPC3 pancreatic cancer model that has K-ras, but has a poor therapeutic effect against a MiaPaCa-2 pancreatic cancer model with mutant K-ras. To increase the therapeutic effect of PH-427 against the MiaPaCa-2 pancreatic cancer model with mutant K-ras, we encapsulated PH-427 into poly(lactic-co-glycolic acid) nanoparticles (PNP) to form drug-loaded PH-427-PNP. PH-427 showed a biphasic release from PH-427-PNP over 30 days during studies in sodium phosphate buffer, and in vitro studies revealed that the PNP was rapidly internalized into MiaPaCa-2 tumor cells, suggesting that PNP can improve PH-427 delivery into cells harboring mutant K-ras. In vivo studies of an orthotopic MiaPaCa-2 pancreatic cancer model showed reduced tumor load with PH-427-PNP as compared with treatment using PH-427 alone or with no treatment. Ex vivo studies confirmed the in vivo results, suggesting that PNP can improve drug delivery to pancreatic cancer harboring mutant K-ras.

Nanoparticle encapsulation and controlled release of a hydrophobic kinase inhibitor: Three stage mathematical modeling and parametric analysis

A mathematical model of drug release that incorporates the simultaneous contributions of initial burst, nanoparticle degradation-relaxation and diffusion was developed and used to effectively describe the release of a kinase inhibitor and anticancer drug, PHT-427. The encapsulation of this drug into PLGA nanoparticles was performed by following the single emulsion-solvent evaporation technique and the release was determined in phosphate buffer pH 7.4 at 37 °C. The size of nanoparticles was obtained in a range of 162-254 nm. The experimental release profiles showed three well defined phases: an initial fast drug release, followed by a nanoparticle degradation-relaxation slower release and then a diffusion release phase. The effects of the controlled release most relevant parameters such as drug diffusivity, initial burst constant, nanoparticle degradation-relaxation constant, and the time to achieve a maximum rate of drug release were evaluated by a parametrical analysis. The theoretical release studies were corroborated experimentally by evaluating the cytotoxicity effectiveness of the inhibitor AKT/PDK1 loaded nanoparticles over BxPC-3 pancreatic cancer cells in vitro. These studies show that the encapsulated inhibitor AKT/PDK1 in the nanoparticles is more accessible and thus more effective when compared with the drug alone, indicating their potential use in chemotherapeutic applications.

Breakthrough performance of large proteins on ion‐exchange membrane columns

Protein adsorption of large proteins on ion‐exchange membrane columns was theoretically and experimentally investigated using batch and fixed‐bed systems. Thyroglobulin was used as the model protein. The study strongly suggests that part of the protein is physically retained inside the column during frontal mode operation. These experimental results were used to obtain a filtration function of the chromatographic system. In the theoretical analysis of the frontal protein adsorption, a model was integrated by the serial coupling of the membrane‐transport model, the filtration model and the system‐dispersion model. Two different techniques were employed in the estimation of the maximum adsorption capacity, the equilibrium desorption constant and the forward interaction rate constant, which are the parameters of the membrane‐transport model. The fit of the model to the experimental data was not possible using the equilibrium parameters obtained in the batch experiments. The parameter estimation using a simplex optimization routine coupled to the solution of the partial differential model equations yields full prediction of the adsorption phenomena.

Systematic evaluation of pH and thermoresponsive poly(n-isopropylacrylamide-chitosan-fluorescein) microgel

Abstract The interesting properties of stimuli-responsive polymers lead to a wide range of possibilities in design and engineering of functional material for the biomedical application. A systematic approach focused on the evaluation of the physical properties of multiresponse (pH and temperature) PNIPAM was reported in this work. The effect of three different molar ratios of poly(n-isopropylacrylamide): chitosan (1:49, 1:99 and 1:198) were evaluated and labeled correspondingly as PC1F, PC2F, and PC3F. An increase in the lower critical solution temperature (LCST) of sample PC1F (34°C) was observed by differential scanning calorimetry (DSC). The presence of low molecular weight chitosan (LMWC) full-interpenetrating polymer (Full-IPN) segments in poly(n-isopropylacrylamide) was confirmed by Fourier-transform infrared spectroscopy (FT-IR). The hydrogel’s water capture was analyzed by two models of swelling, the power law model and a model that considers the relaxation of polymeric chains of the hydrogel, finding good correlations with experimental data in both cases. Sample PC3F resulted with higher swellability, increasing the weight of the hydrogel around seven times. Hydrogel pH-sensibility was confirmed placing the samples at different pH environments, with an apparent increase in swellability for acidic conditions, confirming the highest swellability for sample PC3F, due to hydrogen bonds boosted by chitosan high molar ratio. Based on these results, the hydrogel obtained has potential as a thermo-pH triggered hydrogel in drug delivery applications.

Methylene Blue Loaded PLGA Nanoparticles: Combined Emulsion, Drug Release Analysis and Photodynamic Activity

The use of polymeric nanoparticles for the control release of photosensitizer compounds such as methylene blue represents a promising option for cancer treatment. Methylene blue (MB) has been of great interest in many areas of clinical medicine, from neurological disorders to cancer chemotherapy [12]. It can be used in photodynamic therapy, which consists on the application of MB in the area of interest, and then activated by light at 665 nm producing reactive oxygen species that leads to the death of the target cell via oxidative damage. Polymeric nanoparticles loaded with methylene blue (MB-PNP) were prepared by using a combined emulsification technique [4-5]. Briefly, MB is dissolved in deionized water and into a DCM solution containing MB and PLGA. The mixture is emulsified at 22% of amplitude (26.5 μm) by sonication. Next, an aqueous solution of 5% w/v PVA is added into the mixture and a second emulsification is carried during at 75% amplitude (90 μm). The solvent is evaporated under magnetic stirring, at room temperature. MB-PNPs are washed by three centrifugation cycles and freeze-dried for further characterization. All experiments were performed by triplicate.

Evaluation of a combined emulsion process to encapsulate methylene blue into PLGA nanoparticles

The delivery of photosensitizer compounds using biodegradable nanoparticles could improve dosage, controlled release and its bioavailability. In this study, methylene blue (MB) loaded PLGA nanoparticles (MB-PNP) are prepared by a new approach combining single and double emulsification techniques. Comparisons of MB-PNP obtained with the combined and the individual techniques are presented. Nanoparticles are characterized by dynamic light scattering, laser Doppler electrophoresis and scanning electron microscopy. Particles prepared by the combined technique presented hydrodynamic diameters of 186 nm. The sizes of MB-PNP obtained from the single emulsion technique are similar to the combined technique, while the diameter of particles prepared by double emulsion increased from 201 nm to 287 nm as the TDL increased. MB-PNP displayed an average zeta potential between −21 mV and −28 mV for all formulations. MB loading ranges between 0.3–1.4%, while the encapsulation efficiency ranges from 8–14%, both depending on the TDL and the preparation technique. In vitro release studies show a monophasic release profile that was analyzed by considering the mechanisms of initial burst, drug diffusion and a combination of them. Experimental results could be better described using a mathematical model of release that simultaneously combines the mechanisms of initial burst and drug diffusion. The approach presented to encapsulate MB and also to analyze the drug release could be extended to other drugs with partial solubility.

Poly(N-isopropylacrylamide)-coated gold nanorods mediated by thiolated chitosan layer: thermo-pH responsiveness and optical properties

Abstract A core-shell of colloidal metal-responsive polymer provides an innovative model in functional materials. These core-shell nanocomposites offer the possibility to control some properties, such as particle size, surface plasmon resonance and morphology. In this research, we demonstrate the successful synthesis and functionality of gold nanorods (GNR) coated with the polymers chitosan (Ch) and poly(N-isopropylacrylamide) (PNIPAM). The polymer coatings are performed using a two-step method. First, GNR were coated with a thiolated chitosan (GNR-Ch) by replacing hexadecyltrimethylammonium bromide with a chitosan thiomer. Structural modification of GNR-Ch was monitored by Fourier transform infrared spectroscopy. Then a second polymeric coating was done by in situ free radical polymerization of N-isopropylacrylamide (NIPAM) on GNR-Ch to obtain the nanocomposite GNR-Ch-PNIPAM. The nanocomposite average size was analyzed by dynamic light scattering. The evolution of ζ potentials during the coatings was measured using electrophoretic mobility. GNR-Ch-PNIPAM presented a collapsed structure when heated above the lower critical solution temperature. The particle size of GNR-Ch-PNIPAM was manipulated by changing the pH. Plasmonic properties were evaluated by UV-Vis spectroscopy. Results showed an important blue shift due to the PNIPAM coating thickness. Thermo- and pH-responsive properties of the nanocomposite GNR-Ch-PNIPAM could be used as a drug delivery system.

Abstract 3752: Nanoparticles delivery of a novel AKT/PDK1 inhibitor inhibits pancreatic cancer tumor growth

AKT (protein kinase B), a pleckstrin homology (PH) lipid binding domain and a serine/threonine kinase containing protein is a key component of the phophatidylinositol-3-kinase (PtdIns3-K) cell survival signaling pathway which is activated in pancreatic cancers. In this study, we describe the effects of a novel inhibitor of AKT/PDK1 (PH-427) that binds to the PH domain of AKT (K i = 2.67 ± 0.37 μM) and PDK1 (K i = 5.20 ± 0.45 μM) [1] thus preventing its binding to PtIns-(3,4,5)P 3 at the plasma membrane and subsequent activation of both kinases. PH-427 inhibits AKT/PDK1 activities at low micromolar concentrations in BxPC-3 (wt-KRAS) and not in MiaPaCa2 (mt-KRAS) human pancreatic cancer cell lines. Accordingly, we have shown that in vivo, PH-427 is poorly efficient in MiaPaCa2 xenografts as compared to BxPC-3 xenografts. In order to increase the potency and delivery of PH-427 to the tumor, we have encapsulated the compound (>10% of the compound) into (poly-d, l-lactide-co-glycolide) (PLGA) nanoparticles (PNP). We demonstrate that PH-427 incorporates well in the nanoparticles (PH-427-PNP) ( 3 ). In conclusion, PH-427 alone did not alter tumor growth in MiaPaCa-2-Luc but was able to reduce tumor growth significantly when encapsulated in PNP. Finally and interestingly, metastasis to the liver and other organs was also significantly inhibited by PH-427-PNP as compared to PNP or PH-427 alone. [1]. Meuillet EJ, Ihle N, Lemos R, Moses SA, Song Z, Zhang S, Mash EA, Powis G (2010). Mol Cancer Ther 9(3):706-717. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3752. doi:1538-7445.AM2012-3752