Nanoengineering of the botulinum neurotoxin serotype a (BoNT/A) to rescue the ischemic brain

Abstract

Stroke is the second leading cause of death worldwide, thus being a disorder that is an ever-growing problem. The most prevalent type of stroke, the ischemic stroke (IS), activates various signaling molecular pathways that ultimately lead to cell death. In fact, the most contributing event for the latter is glutamate excitotoxicity. The currently available therapies are based on the reperfusion of the blood flow to the brain, lacking neuroprotective or neuroregenerative properties, essential for improving IS outcomes. Therefore, there is an urgent need for the development of novel therapies. Botulinum neurotoxins (BoNTs), coupled with Nanomedicine, could provide an answer to achieve this need. Their light chain (LC) specifically cleaves the proteins responsible for mediating neurotransmitter release, namely glutamate, enabling the decrease of excitotoxicity. BoNTs remain largely unexplored for application in the CNS, due to their inherent toxicity, that should be controlled to prevent undesired effects. Nanotechnology arises as a promising solution to tackle the challenge of delivery these neurotoxins, coupling the ability to encapsulate the LC with the possibility of having a targeted delivery. Hence, this work aimed at the production and purification of the active BoNT/A-LC and posteriorly, the formation of complexes (DETOX) with a fully biodegradable dendrimer (fbA). This nanodelivery system, produced and patented by our lab, is expected to enable DETOX to reach ischemic neurons, the therapeutic target. In this work, the production of the LC protein was accomplished through bacterial expression vectors and by following an optimized purification workflow we achieved a high degree of purity of the recombinant protein (> 99%). The production of the biologically active LC was demonstrated by the detection of the cleaved SNAP-25 (cSNAP-25), the catalytic target of BoNT/A, in a culture of primary cortical neurons. Then, we encapsulated the LC protein in the fbA dendrimer, having tested different molar ratios. By particle size determination techniques, the formation of DETOX seemed more promising for the 20:1 and 30:1 molar ratios of fbA/LC. Finally, the transfection of DETOX with primary cortical neurons did not show an increase in the detection of cSNAP-25, in comparison to the LC only, however several optimizations still need to be tested. Nevertheless, we were able to demonstrate that the LC protein maintains its biological activity when encapsulated within the fbA dendrimer. Despite the validations that need to be performed, these results indicate the formation of a promising nanosystem for the delivery of the BoNT/A-LC in neurons. Furthermore, these findings indicate an advancement for the application of these neurotoxins to the CNS, and in the use of dendrimers as delivery systems of proteins.

Stroke is the second leading cause of death worldwide, thus being a disorder that is an ever-growing problem. The most prevalent type of stroke, the ischemic stroke (IS), activates various signaling molecular pathways that ultimately lead to cell death. In fact, the most contributing event for the latter is glutamate excitotoxicity. The currently available therapies are based on the reperfusion of the blood flow to the brain, lacking neuroprotective or neuroregenerative properties, essential for improving IS outcomes. Therefore, there is an urgent need for the development of novel therapies. Botulinum neurotoxins (BoNTs), coupled with Nanomedicine, could provide an answer to achieve this need. Their light chain (LC) specifically cleaves the proteins responsible for mediating neurotransmitter release, namely glutamate, enabling the decrease of excitotoxicity. BoNTs remain largely unexplored for application in the CNS, due to their inherent toxicity, that should be controlled to prevent undesired effects. Nanotechnology arises as a promising solution to tackle the challenge of delivery these neurotoxins, coupling the ability to encapsulate the LC with the possibility of having a targeted delivery. Hence, this work aimed at the production and purification of the active BoNT/A-LC and posteriorly, the formation of complexes (DETOX) with a fully biodegradable dendrimer (fbA). This nanodelivery system, produced and patented by our lab, is expected to enable DETOX to reach ischemic neurons, the therapeutic target. In this work, the production of the LC protein was accomplished through bacterial expression vectors and by following an optimized purification workflow we achieved a high degree of purity of the recombinant protein (> 99%). The production of the biologically active LC was demonstrated by the detection of the cleaved SNAP-25 (cSNAP-25), the catalytic target of BoNT/A, in a culture of primary cortical neurons. Then, we encapsulated the LC protein in the fbA dendrimer, having tested different molar ratios. By particle size determination techniques, the formation of DETOX seemed more promising for the 20:1 and 30:1 molar ratios of fbA/LC. Finally, the transfection of DETOX with primary cortical neurons did not show an increase in the detection of cSNAP-25, in comparison to the LC only, however several optimizations still need to be tested. Nevertheless, we were able to demonstrate that the LC protein maintains its biological activity when encapsulated within the fbA dendrimer. Despite the validations that need to be performed, these results indicate the formation of a promising nanosystem for the delivery of the BoNT/A-LC in neurons. Furthermore, these findings indicate an advancement for the application of these neurotoxins to the CNS, and in the use of dendrimers as delivery systems of proteins.