| Spinal cord injuries (SCI) afflict millions of people every year worldwide. Since no cure exists for such neurotraumatic episodes, patients thus experience highly debilitating symptoms. These comprehend partial to complete muscular weakness and sensory loss, and commonly lead to paraplegia or tetraplegia. The tremendous complexity of cellular and biochemical reactions that follow the initial trauma, along with the lack of specificity of the drugs used, are hindering the development of effective treatments for SCI. Current clinical options are thus mainly based on palliative care, and systemic high dose administration of methylprednisolone (MP), a powerful anti-inflammatory and antioxidant corticosteroid that has shown to lead to functional recovery. However, the unspecificity and deleterious side effects of MP are making some clinicians cautious towards its administration. Actually, this treatment is very controversial, therefore, new enhanced approaches are needed to repair the injured spine and provide SCI patients a better quality of life. The most recent developments in nanotechnology show great promise for tissue engineering advanced solutions. Recently, poly(amido)amine (PAMAM) dendrimer nanoparticles grafted with CMCht have been proposed as intracellular nanocarriers for stem cell osteogenic differentiation. In this thesis, it was explored the application of the developed CMCht/PAMAM dendrimer nanoparticles for neuroprotection of the injured spinal cord tissue, ultimately contributing to regeneration and repair. Firstly, functionalization of CMCht/PAMAM dendrimer nanoparticles was performed covalently binding an antibody to its structure, and then incorporating a relevant drug, such as MP. Both modifications were confirmed using spectroscopic techniques and in vitro biological evaluation was performed in primary cortical glial cultures. No cytotoxicity associated with the multifunctional CMCht/PAMAM dendrimer nanoparticles was observed. In vitro internalization studies revealed a differential uptake, when the antibody was bond to the CMCht/PAMAM dendrimer nanoparticles. Therefore, the addition of targeting moieties to the nanoparticles contributed to a modulation of the nanoparticle uptake by glial cells. In order to analyze the intracellular trafficking, as well as internalization and clearance routes, electrophysiology recordings of live astrocyte (single-cell) membrane capacitance were performed after incubation with MP-loaded CMCht/PAMAM dendrimer nanoparticles. The patch-clamp studies showed that nanoparticles do affect both endocytosis and exocytosis rates. In fact, these observations were further confirmed by confocal microscope visualization of astrocyte endocytotic and exocytotic vesicles. To our knowledge, this study clarified for the first time the endocytotic/exocytotic pathways that nanoparticles follow in primary nervous cells, demonstrating for the first time the exocytotic clearance of the nanoparticles. Following this, administration of FITC-labeled MP-loaded CMCht/PAMAM dendrimer nanoparticles in the cerebrospinal fluid (CSF) of healthy rats was performed. The distribution of the nanoparticles was widespread along several brain areas and layers, showing that once the blood-brain barrier (BBB) is overcome the nanoparticles are easily transported and retained in the brain tissue. Moreover, the drug was shown to be acting intracellularly by protein expression quantification. Subsequently, extensive investigation of the physico-chemical characteristics of the MP-loaded nanoparticles was carried out revealing its spherical 109 nm structure and zeta potential stability. Moreover, a preliminary therapeutic assessment of this system was analyzed in vitro in microglial cultures, and in vivo in an animal model of SCI. Promising results were obtained with the successful modulation of microglia proliferation, and significant locomotor improvements in the treated injured animals. Finnaly, biocompatibility and functionality studies were performed in Schwann cell pure cultures and co-cultures with dorsal root ganglia neurons, when in contact with the MP-loaded CMCht/PAMAM dendrimer nanoparticles. The presence of nanoparticles has not affected the cell morphology or typical distribution in culture. Moreover, extensive myelination was performed by Schwann cells that enveloped the axons of dorsal root ganglia neurons, showing normal viability and function. These results open new possibilities for therapeutic strategies, namely combination of Schwann cell transplantation and nanoparticle administration. From these findings, new knowledge regarding nanoparticle interaction with nervous cells was obtained, along with the potential therapeutic value. For the first time, multifunctionalization of CMCh/PAMAM dendrimer nanoparticles was successfully accomplished and proved to modulate the cell uptake rates. Moreover, the innovative use of patch-clamp electrophysiology for the quantification of vesicle formation/fusion in the cell membrane following nanoparticle incubation proved to be useful in this type of studies. The promising locomotor improvements observed in nanoparticle-treated SCI animals, along with the biocompatibility of these nanoparticles for CNS applications, bring new hopes for the development of successful strategies for SCI repair. |
