Shannon Ewanick

A Scalable, Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA

No HeadingPurpose.A fully scalable and extrusion-free method was developed to prepare rapidly and reproducibly stabilized plasmid lipid particles (SPLP) for nonviral, systemic gene therapy.Methods.Liposomes encapsulating plasmid DNA were formed instantaneously by mixing lipids dissolved in ethanol with an aqueous solution of DNA in a controlled, stepwise manner. Combining DNA-buffer and lipid-ethanol flow streams in a T-shaped mixing chamber resulted in instantaneous dilution of ethanol below the concentration required to support lipid solubility. The resulting DNA-containing liposomes were further stabilized by a second stepwise dilution.Results.Using this method, monodisperse vesicles were prepared with particle sizes less than 200 nm and DNA encapsulation efficiencies greater than 80%. In mice possessing Neuro 2a tumors, SPLP demonstrated a 13 h circulation half-life in vivo, good tumor accumulation and gene expression profiles similar to SPLP previously prepared by detergent dialysis. Cryo transmission electron microscopy analysis showed that SPLP prepared by stepwise ethanol dilution were a mixed population of unilamellar, bilamellar, and oligolamellar vesicles. Vesicles of similar lipid composition, prepared without DNA, were also <200 nm but were predominantly bilamellar with unusual elongate d morphologies, suggesting that the plasmid particle affects the morphology of the encapsulating liposome. A similar approach was used to prepare neutral egg phosphatidylcholine:cholesterol (EPC:Chol) liposomes possessing a pH gradient, which was confirmed by the uptake of the lipophilic cation safranin O.Conclusions.This new method will enable the scale-up and manufacture of SPLP required for preclinical and clinical studies. Additionally, this method now allows for the acceleration of SPLP formulation development, enabling the rapid development and evaluation of novel carrier systems.

The effect of biomass moisture content on bioethanol yields from steam pretreated switchgrass and sugarcane bagasse

This study aimed to determine the effect of moisture content of three different feedstocks on overall ethanol yield. Switchgrass and sugarcane bagasse from two sources were either soaked in water (∼80% moisture) or left dry (∼12% moisture), and half each of these were impregnated with 3%w/w SO(2) and all were steam pretreated. The twelve resulting substrates were compared based on overall sugar recovery after pretreatment, cellulose conversion following enzymatic hydrolysis, and ethanol yield following simultaneous saccharification and fermentation. The overall ethanol yield after simultaneous saccharification and fermentation of hexoses was 18-28% higher in samples that were soaked prior to SO(2) addition than in SO(2)-catalyzed samples that were not soaked. In samples that were uncatalyzed, soaking made little difference, indicating that the positive effect of increased moisture content may be related to increased permeability of the biomass to SO(2).

Comparison of methods to assess the enzyme accessibility and hydrolysis of pretreated lignocellulosic substrates

Fiber size analysis, water retention value, and Simons’ stain measurements were assessed for their potential to predict the susceptibility of a given substrate to enzymatic hydrolysis. Slight modifications were made to the fiber size analysis and water retention protocols to adapt these measurements to evaluate substrates for cellulolytic hydrolysis rather than pulps for papermaking. Lodgepole pine was pretreated by the steam and ethanol-organosolv processes under varying conditions. The Simons’ stain procedure proved to be an effective method for indicating the potential ease of enzymatic hydrolysis of substrates pretreated by either process or when the pretreatment conditions were altered.

Real-time understanding of lignocellulosic bioethanol fermentation by Raman spectroscopy

BackgroundA substantial barrier to commercialization of lignocellulosic ethanol production is a lack of process specific sensors and associated control strategies that are essential for economic viability. Current sensors and analytical techniques require lengthy offline analysis or are easily fouled in situ. Raman spectroscopy has the potential to continuously monitor fermentation reactants and products, maximizing efficiency and allowing for improved process control.ResultsIn this paper we show that glucose and ethanol in a lignocellulosic fermentation can be accurately monitored by a 785 nm Raman spectroscopy instrument and novel immersion probe, even in the presence of an elevated background thought to be caused by lignin-derived compounds. Chemometric techniques were used to reduce the background before generating calibration models for glucose and ethanol concentration. The models show very good correlation between the real-time Raman spectra and the offline HPLC validation.ConclusionsOur results show that the changing ethanol and glucose concentrations during lignocellulosic fermentation processes can be monitored in real-time, allowing for optimization and control of large scale bioconversion processes.

Hydrothermal pretreatment of lignocellulosic biomass

Abstract: Lignocellulosic biomass has long been recognized as a potential sustainable source of mixed sugars fermentation to biofuels and other biochemicals. The hydrothermal pretreatments (steam explosion and hot water pretreatment) are the most effective pretreatments for a variety of biomass types and have been shown to work effectively at a commercial scale. Here, we consider the technical maturity of the hydrothermal pretreatments by looking at the process history, describing the mode of reactions and analyzing the influence of pretreatment conditions on the physico-chemical properties of pretreated biomass. Finally, we compare the effectiveness of hydrothermal pretreatments and outline the remaining challenges associated with harnessing the pretreatment for production of biochemicals.

Post-treatment mechanical refining as a method to improve overall sugar recovery of steam pretreated hybrid poplar.

This study investigates the effect of mechanical refining to improve the sugar yield from biomass processed under a wide range of steam pretreatment conditions. Hybrid poplar chips were steam pretreated using six different conditions with or without SO2. The resulting water insoluble fractions were subjected to mechanical refining. After refining, poplar pretreated at 205°C for 10min without SO2 obtained a 32% improvement in enzymatic hydrolysis and achieved similar overall monomeric sugar recovery (539kg/tonne) to samples pretreated with SO2. Refining did not improve hydrolyzability of samples pretreated at more severe conditions, nor did it improve the overall sugar recovery. By maximizing overall sugar recovery, refining could partially decouple the pretreatment from other unit operations, and enable the use of low temperature, non-sulfur pretreatment conditions. The study demonstrates the possibility of using post-treatment refining to accommodate potential pretreatment process upsets without sacrificing sugar yields.

Use of Raman spectroscopy for continuous monitoring and control of lignocellulosic biorefinery processes.

Abstract The production of fuels and chemicals from lignocellulosic biomass demands efficient processes to compete with fossil fuel-derived products. Key biorefinery processes, such as enzymatic hydrolysis of cellulose and microbial fermentation, can be monitored by advanced sensors in real time, providing information about reactant and product concentration, contamination, and reaction progress. Spectroscopic techniques such as Raman spectroscopy provide a means of quickly and accurately assessing many types of reaction mixtures non-destructively, in real time, and with no costly sample preparation and analysis time. Raman spectroscopy techniques have been developed to accurately quantify a number of compounds present in lignocellulosic processes, and methods have been developed to overcome the presence of fluorescent compounds that can increase the spectral background. Online Raman sensors also can provide the feedback measurements necessary for advanced process controls (APCs). Specifically, model predictive control, a common APC used extensively throughout similar processing industries, is especially well suited for ensuring optimal production of bio-based chemicals from lignocellulosic material.