Nanotechnologies for CNS drug delivery: therapy for the neurological compartment in the Mucopolisaccharidoses

Lysosomal storage disorders (LSDs) are a group of neurometabolic syndromes, mostly due to the deficit of one lysosomal enzyme. Many LSDs affect most of the organ systems and about two-thirds of the patients also present neurological and cognitive impairment. Enzyme replacement therapy, the most common therapeutic strategy applied to some LSDs, although determining some clinical improvements, has revealed to be ineffective on the CNS disease, due to enzymes' inability to cross the blood-brain barrier (BBB). So alternative methods to achieve transcytosis into the CNS need to be explored. Among LSDs, Mucopolysaccharidoses (MPS) are characterized by a totally or partially defective activity of lysosomal enzymes involved in the catabolism of the glycosaminoglycans (GAGs), which, therefore, heavily accumulate within cellular compartment and in the extracellular matrix. In this study, in which MPS type I and type II have been in particular evaluated, polymeric nanoparticles (NPs), modified with a glycopeptide of 7 amino acids (g7), already tested for the delivery of low molecular weight molecules, were tested as possible vehicle for BBB crossing and delivering of the therapeutic recombinant enzymes to the CNS after systemic administration in mice. Results obtained in preliminary studies in the MPS I and MPS II mouse models, clearly showed that g7-NPs are able to deliver the model drug FITC-albumin to the brain, by crossing the BBB, in all treated mice (Idua-ko, Ids-ko and wt). The subsequent in vivo preliminary study in the MPS II mouse model, using g7-NPs loaded with the recombinant IDS enzyme and labelled with Rhodamine B (g7-NPs-IDS-R), confirmed that g7-NPs can transport the enzyme through the BBB and paved the way for the upcoming in vitro and in vivo efficacy studies. In the in vitro study fibroblasts obtained from MPS II patients were treated with untargeted NPs (devoid of g7), loaded with the recombinant enzyme for MPS II. Data obtained suggested that nanoparticles may need more than 2 weeks to open up and release the recombinant enzyme or, otherwise, that they may need a more complex physiological system with respect to the in vitro one. To evaluate transfer efficiency and the enzymatic activity induced by g7-NPs loaded with the recombinant enzyme (g7-NPs-IDS), an in vivo pilot study in the MPS II mouse model was conducted. Mice were treated with a single dose of enzyme and sacrificed after 7 or 14 days. No induced IDS activity or reduction in GAG storage was, however, detected in liver and brain of g7-NPs-IDS treated mice. Although the results obtained in the in vitro and in vivo pilot studies were mostly negative, it was important to carry out a medium-term analysis to assess the possible opening of NPs in a longer time. To this aim, an in vivo study was conducted in the MPS II mouse model, by treating mice once a week for 6 weeks. Biochemical, histological, immunohistochemical and immunofluorescence evaluations, conducted in liver and brain of g7-NPs-IDS treated mice suggested that a period of 6 weeks is again too short to detect the opening of the NPs and the release of the encapsulated enzyme. However, results obtained from this medium-term study are encouraging as they show a slight trend towards improvement in brain and liver of g7-NPs-IDS treated mice. Further studies, at the moment ongoing, are needed to understand the timing of release of the enzyme from the NPs; in addition, we are re-formulating the nanoparticles with the aim to maintain the same transport efficiency to the CNS, although allowing a significant reduction of the timing of drug release.