Resumen de Advanced methodology for LPS capture from biofluids
- español
El lipopolisacárido (LPS), o endotoxina, es el principal componente de la membrana externa de las bacterias Gram negativas donde el lípido A es el segmento responsable de su toxicidad. Su presencia supone un grave riesgo tanto en diferentes industrias y entornos como cuando llega al torrente sanguíneo pudiendo conducir a la sepsis, una respuesta exagerada al LPS que desencadena una supresión inmunitaria, una disfunción orgánica o incluso la muerte. Lamentablemente, los métodos alternativos para la eliminación de contaminantes a través de procesos de detoxificación extracorpórea de la sangre presentan inconvenientes que hacen que la detección/eliminación de endotoxinas sea un reto crucial para lograr procesos de detoxificación seguros y eficaces. En este sentido, los dispositivos magnetofluídicos merecen especial atención e implican dos etapas principales; el secuestro de LPS en nanopartículas magnéticas (MNPs) convenientemente funcionalizadas y, la eliminación del complejo MNPs-LPS del fluido biológico. En consecuencia, esta disertación aporta una metodología integrada para avanzar en el diseño de la etapa de secuestro de LPS para promover su separación de los biofluidos a través de la síntesis de una proteína antilipopolisacáridos (LALF) procedente de la especie Limulus polyphemus mediante técnicas de ingeniería genética además de la cuantificación de la fuerza de unión de la proteína LALF al LPS mediante un nuevo enfoque y teniendo en cuenta las variables que afectan a la formación del complejo. Además, con el objeto de contribuir al desarrollo de una aplicación para la captura de LPS en continuo, como primera aproximación, se aborda para la separación homogénea y heterogénea L-L de aniones acuosos (cromato) en microdispositivos experimentalmente y mediante un modelo teórico desarrollado con ANSYS FLUENT, sentando las bases para continuar con el diseño microfluidico para la separación L-S y finalmente, su aplicación a la captura de LPS.
- español
Lipopolysaccharide (LPS), also called endotoxins, is the major component of the outer membrane of Gram-negative bacteria and is constituted of three regions; the O-specific chain, the core region and the lipid A, which is the responsible segment of toxicity. Lipid A presence often poses a serious risk not only when delivered in the bloodstream but also in several industrial fields.
As described in chapter 1, endotoxin contamination has been reported in different industries and environments as for example, in water and sewage treatment plants and in the cotton, food and pharmaceutical industry. Also, endotoxins have also been detected in house-dust, in bioaerosols, soil, water, air conditioners and waste treatment plants where organic-water solvent extraction systems, ultrafiltration processes and chromatographic techniques have been employed to avoid contamination in both processes and products.
Besides, LPS is highly toxic when is present in human blood, and causes fever, physical discomfort, leukocyte alterations and respiratory affections. In the worst scenario it can lead to sepsis, an exaggerated response to LPS that triggers immune suppression, organ dysfunction or even death. Despite the advances in knowledge on sepsis pathophysiology, several observational studies and clinical trials have failed to identify effective adjuvant therapies that could modify the course of the disease.
In the search of alternative methods of contaminant removal, blood cleansing procedures for the extracorporeal endotoxin separation have received increased attention. In this context, various strategies for LPS separation from contaminated fluids have been developed such as organic solvent extraction, the use of detergents such as Triton X-100 or antibiotics (polymyxin B) immobilized on polystyrene fibers and packed into columns ready for direct perfusion of the biofluids. Unfortunately, in spite of these efforts, most of these systems have drawbacks that make endotoxin detection/removal a crucial challenge to achieve safe and effective detoxification processes.
In this regard, progress and capabilities of magnetofluidic devices deserves special attention. Magnetofluidic devices entail two main stages taking part in the whole process; the initial entrapment of LPS in conveniently functionalized magnetic nanoparticles (MNPs) and, the removal of the loaded MNPs from the biofluid. Whereas the second stage has received the attention of a great number of researchers, the LPS capture, where functionalized beads selectively bind to the target pathogen needs further research.
Consequently, this dissertation reports the methodology to advance in the design of the LPS sequestration stage to promote its separation from biofluids. To this end, first, chapter 2 reports the procedure for an antilipopolysaccharides protein from Limulus polyphemus (LALF) synthesis based on genetic engineering techniques where the first step was to assembly a plasmid, a small, circular, double-stranded DNA molecule consisting of a gene encoding the protein of interest in a specialized vehicle called vector. Subsequently, the circular DNA was transformed into cells capable of expressing the protein, which, in a final stage was successfully purified.
Afterwards, chapter 3 addresses the binding strength of the LALF protein to LPS quantification through a newly approach that consisted of a functionalization stage where the protein was supported on the surface of agarose beads and then, a capture stage where the decorated particles were contacted to fluorescent LPS solution. Moreover, variables affecting the beads-LALF-LPS complex formation such as binding and capture temperature, the optimum bead: protein and protein:LPS ratios, were experimentally studied to accurately determine the LALF activity.
Once LALF:LPS complexation equilibrium was determined, it was necessary to develop an application to carry out the continuous LPS capture aimed at fluid detoxification based on the use of flow-through microdevices. Because of the novelty of this approach, an in-depth methodology has been developed and described in chapter 3, making use of chemical systems with known equilibrium and kinetics and maintaining the fluid-dynamic similarity. Thus, the design of microdevices for the homogeneous and L-L heterogeneous separation of aqueous anions (chromate) has been developed, setting the grounds to continue with the microfluidic design of L-S separation and finally its application to LPS capture.
ANSYS FLUENT software was used to develop a flexible model that solves under dynamic conditions both Navier-Stokes and species balance equations; the model also implements the surface tension between the liquid phases that had been experimentally determined, and the fluid-wall interaction through the measurement of the contact angle.
Last, experimental and simulated results were compared in order to validate the model and apply it to the subsequent analysis of the reactive L-S systems and, finally perform the capture of LPS.
