| Summary: | Lysosomal storage diseases (LSD) comprise a family of genetic disorders affecting roughly 1 in 5000 births. Storage disorders involving the central nervous system (CNS) present unique challenges to researchers and medical practitioners. Although current treatment strategies including enzyme replacement therapy and gene therapy have proven effective for forms of the disease not involving the CNS, there is currently no effective way of delivering proteins to the CNS without direct injection. Putative reasons for this include difficulty of protein delivery across the blood brain barrier and protein targeting to specific cell types. This presents additional challenges for lysosomal storage disorders such as late infantile neuronal ceroid lipofuscinosis (LINCL), a lysosomal storage disorder in which a mutation in Batten disease (CLN2, 11p15) causes neurons to fail to produce enzymatically viable tripeptidylpeptidase 1 (TPP1) leading to substrate accumulation in the developing brain and ultimately death of neurons.
Our lab has previously shown that CdSe/ZnS (core/shell) luminescent semiconductor nanocrystals or quantum dots (QDs) coated with polyethylene glycol (PEG)-appended dihydrolipoic acid (DHLA) can bind peptide JB577 (AcWG(Pal)VKIKKP9GGH 6) through the histidine residues on the peptide. The coating on the QD provides colloidal stability and this peptide complex uniquely allows the QDs to be taken up by cultured cells and readily exit the endosome into the soma. Here we show that use of a polyampholyte coating (in which the neutral PEG is replaced by the negatively heterocharged compact ligand), results in the specific targeting of the peptide to neurons in mature rat hippocampal slice cultures. There was no noticeable uptake by astrocytes, oligodendrocytes or microglia (identified by immunocytochemistry), demonstrating neuronal specificity to the overall negatively charged CL4 coating. In addition, EM (electron microscopy) images confirm the endosomal egress ability of the JB577 peptide shows a much more disperse cytosolic distribution of the CL4 QDs conjugated to JB577 compared with CL4 QDs alone. This suggests a novel and robust way of delivering neurotherapeutics to neurons.
Despite our extensive knowledge of the structure of negatively charged cell surface proteoglycans and sialoglycoconjugates in the brain, we have little understanding of how their negative charge contributes to brain function. We compared three polymer coatings giving an overall charge of positive (PEG600), zwitterionic neutral (CL1) and more negatively zwitterionic (CL4, CL2) for their ability to deliver a JB577 to the brain and confirmed both the preferential uptake associated with negative surface charge and the lack of uptake by glial cells. On the other hand, the positively charged QDs seemed to exhibit the inverse effect, targeting oligodendrocytes with a reduction in neuronal targeting. The role of the negatively charged proteoglycan extracellular matrix (ECM) in restricting uptake was also investigated by digesting the neonatal rat hippocampal slices with chondroitinase ABC. This showed uptake of QDs by oligodendrocytes, but not by astrocytes or microglia. Finally, using a similar approach, we then show that a recombinant His6-tagged GFP as well as lysosomal enzyme (TPP1) could be delivered to neurons, suggesting that QD delivery could be a useful approach to enzyme replacement therapy in neuronal ceroid lipofuscinoses.
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