Desarrollo y caracterización de un conjugado del péptido análogo a glucagón (GLP-1) a nanopartículas de oro, como nueva forma de entrega de biomoléculas con potencial aplicación en diabetes

Para probar este concepto, se sintetizaron en fase sólida tres nuevos análogos del GLP-1, los cuales fueron caracterizados por cromatografía líquida y espectrometría de masas. Adicionalmente, a los péptidos se les evaluó su estructura secundaria por dicroísmo circular y su actividad insulinotrópica in vitro en la línea celular de páncreas Beta-TC-6. Posteriormente se prepararon y caracterizaron fisicoquímicamente los conjugados obtenidos; para ello se utilizaron técnicas de espectrofotometría UV-visible, microscopía electrónica de transmisión, potencial zeta y dispersión dinámica de la luz. Finalmente se evaluó la estabilidad de los conjugados, se estudió su permeabilidad intestinal, utilizando un sistema in vitro basado en un cocultivo de células de adenocarcinoma de colon humano y mucosecretoras (Caco-2/goblet), y además se determinó la actividad hipoglicemiante in vivo de uno de los péptidos y su respectivo conjugado a AuNP, después de una administración intraperitoneal en ratas. Los resultados obtenidos revelaron que los tres péptidos sintetizados: GLP-1(7- 37)-Lys(Acet), GLP-1(7-37)-Lys(Cys) y GLP-1(7-37)-Lys(PegCys), tienen una conformación de tipo α hélice en disolución acuosa y presentan una actividad insulinotrópica similar a la de la incretina endógena GLP-1. Sin embargo, generan conjugados que difieren en estabilidad y capacidad de penetrar el epitelio intestinal, probablemente debido al tipo de interacción que generan con las AuNP. De esta forma, los conjugados de los péptidos que contienen cisteína (péptidos tiolados) resultaron ser más estables que el del péptido sin cisteína, frente al pH gástrico e intestinal, pues generan una interacción de tipo Au-S que le otorga una mayor estabilidad estérica al sistema coloidal. Por otra parte, el conjugado de AuNP al péptido GLP-1(7-37)-Lys(PegCys) fue el que penetró en mayor medida la monocapa de células, encontrándose aproximadamente un 0.016 % de oro después de 4 h de incubación, mientras que con los otros conjugados el nivel de oro no superó el 0.008 %. Además los péptidos y sus conjugados a AuNP no presentaron toxicidad en los sistemas celulares empleados (Beta-TC-6 y Caco-2/goblet, respectivamente). Finalmente, de la evaluación de la actividad hipoglicemiante in vivo se obtuvo que la eficacia hipoglicémica para el análogo GLP-1(7-37)-Lys(PegCys) y su conjugado a AuNP fue similar a la de la incretina endógena (18.7 ± 6.67 %, 27.6 ± 6.33 % y 22.1 ± 8.26 %, respectivamente), revelando que tanto la modificación realizada en el extremo C- terminal de GLP-1, como la presencia de la AuNP, no afectan su bioactividad. De esta forma, el sistema de entrega desarrollado en esta Tesis demostró penetrar las células intestinales en diferentes grados, y disminuir los niveles de glicemia in vivo de una manera similar al péptido endógeno después de su administración intraperitoenal, revelando que este tipo de sistemas es capaz de atravesar barreras biológicas y que la conjugación al nanomaterial no afecta la actividad de la biomolécula, por lo que podría convertirse en una estrategia para la entrega de biomacromoléculas con aplicación en diabetes
 Generally, the therapeutic potential of peptides and proteins is hampered by their physicochemical characteristics, which prevent their successful delivery. In this regard, GLP-1 is a peptide incretin that has great therapeutic usefulness due to their interesting action on glucose metabolism, so it is currently an excellent candidate option for the treatment of the type 2 Diabetes Mellitus. However, as a peptidic macromolecule, GLP- 1 has a poor permeability across the intestinal epithelium and is very susceptible to the enzymatic degradation. To overcome these drawbacks some efforts have been made to improve its therapeutic efficacy, such as development of metabolically stable analogs, but very few studies have been focused on the study of the delivery systems to prolong their action and improve its bioavailability. In this Thesis the study of a potential delivery system based on the use of gold nanoparticles (AuNP) for biomacromolecules, such as GLP-1, is presented. This system searches for to increase the permeability of peptides across the intestinal epithelium and enhance its bioavailability while retaining the biological activity of the biomolecules. Thus, a nanoscale delivery system was developed, wherein GLP-1 analogues were conjugated to the surface of AuNP. To probe this concept, three new analogs of GLP-1 on solid phase were synthesized; the peptides were characterized by liquid chromatography and mass spectrometry and additionally their secondary structure and in vitro insulinotropic activity, using Beta-TC-6 pancreatic cells, were evaluated. Subsequently, the conjugates were physicochemically characterized by UVvisible spectrophotometry, transmission electron microscopy, zeta potential and dynamic light scattering. Finally, the stability of the conjugates and their intestinal permeability, using a mucosecretory in vitro system based on human colon adenocarcinoma cells (Caco-2) and goblet cells were studied. Additionally, the in vivo hypoglycemic activity after intraperitoneal administration en rats of one of the peptides and its conjugate was also determinate. The results revealed that all three peptides synthesized: GLP-1(7-37)-Lys(Acet), GLP-1(7-37)-Lys(Cys) and GLP-1(7-37)-Lys(PegCys), have an α helix conformation in solution, and equal insulinotropic activity than the endogenous incretin GLP-1. However, their conjugates differ in stability and ability to penetrate the intestinal epithelium, probably due to the difference in the interaction with the AuNP. In this way, the conjugates of the peptides containing cysteine (thiolated peptides) were more stable, to the gastric and intestinal pH, than the peptide without cysteine because generate an interaction of Au-S that gives a greater steric stability to the colloidal system. Moreover, the conjugate of GLP-1(7-37)-Lys (PegCys) to AuNP penetrated the cell monolayer in a greater extent than the others, being found about 0.016 % gold after 4 h of incubation, while with the other conjugated the gold level did not exceed 0.008 %. In addition, peptides and their conjugates to AuNP did not show toxicity at cell systems used (Beta-TC-6 and Caco-2/goblet, respectively). Finally, the evaluation of the in vivo hypoglycemic activity showed that the hypoglycemic efficacy for the GLP-1(7-37)-Lys (PegCys) analogue and its AuNP conjugate were similar to the endogenous incretin (18.7 ± 6.67 %, 27.6 ± 6.33 % and 22.1 ± 8.26 %, respectively), revealing that the modification in the C-terminal of the GLP-1 molecule and the presence of the AuNP, do not affect its bioactivity. Thereby, the delivery system developed in this Thesis showed penetrate the intestinal cells in different degrees, and decrease blood glucose levels in vivo in a similar way to the endogenous peptide after intraperitoenal administration, revealing that this type of system is capable of cross biological barriers and that the conjugation to the nanomaterial does not affect the activity of the biomolecule, so it could become a strategy for the delivery of biomacromolecules with application in diabetes