Abstract
BackgroundThe use of siRNA-based gene silencing has been recently underscored as a potential therapeutic strategy for the treatment of neurological disorders. However, the stability of siRNA and other small molecule therapeutics is challenged by their intrinsic instability and limited passage across the blood–brain barrier (BBB). Based on these premises, our objective was to characterize/optimize odorranalectin (OL), a small non-immunogenic lectin-like peptide, as a carrier for targeted delivery across the BBB. For this purpose, 5(6)-carboxyfluorescein-conjugated OL and scramble peptide were synthesized, and then their BBB cellular internalization/trafficking and stability were characterized versus temperature, pH and serum content in the media in hCMEC/D3 cells as a model of BBB endothelium. Specifically, integrity of the internalized peptide in cell lysates was analyzed by LC/MS while cellular distribution and intracellular trafficking of OL was examined by fluorescence microscopy with early-late endosome (pHRodo Red®) and lysosome (Lysotracker®) markers.ResultsOur data show that cellular uptake of OL increased linearly with the concentrations tested in this study at 37 °C and the uptake was two to threefolds higher when compared to scramble peptide. While there were no differences for scramble peptide, the uptake of OL decreased by 50% at 4 °C incubation (vs. 37 °C). No effects of pH were observed on endothelial uptake of OL. Immunofluorescence studies also indicated a significant cellular internalization of OL that remained intact (as evaluated by LC–MS/MS) and co-localized with endosomal, but not lysosome marker. Importantly, OL was found non-toxic to cells at all concentrations tested.ConclusionsIn summary, our data suggest the existence of a receptor-mediated transcytosis pathway for cellular uptake of OL at the BBB endothelium. However, in vivo studies will be needed to assess the siRNA loading capacity of OL and its trans-BBB transport efficiency for targeted delivery in the brain.
Highlights
The use of siRNA-based gene silencing has been recently underscored as a potential therapeutic strategy for the treatment of neurological disorders
Cellular uptake of FAM‐OL peptide in blood–brain barrier (BBB) endothelial cells As shown in Fig. 1b, FAM-OL peptide tested in the concentration range (0–200 μg/mL) did not affect the viability of hCMEC/D3 endothelial cells
Immunofluorescence analysis showed a concentration-dependent increase in cellular uptake of FAM-OL peptide in hCMEC/D3 cells following 3 h incubation at 37 °C (Fig. 2a1–a3)
Summary
The use of siRNA-based gene silencing has been recently underscored as a potential therapeutic strategy for the treatment of neurological disorders. The stability of siRNA and other small molecule therapeutics is challenged by their intrinsic instability and limited passage across the blood–brain barrier (BBB). Based on these premises, our objective was to characterize/optimize odorranalectin (OL), a small non-immunogenic lectin-like peptide, as a carrier for targeted delivery across the BBB. Our objective was to characterize/optimize odorranalectin (OL), a small non-immunogenic lectin-like peptide, as a carrier for targeted delivery across the BBB For this purpose, 5(6)-carboxyfluorescein-conjugated OL and scramble peptide were synthesized, and their BBB cellular internalization/trafficking and stability were characterized versus temperature, pH and serum content in the media in hCMEC/D3 cells as a model of BBB endothelium. No binding was observed with d-glucose, Sajja et al BMC Neurosci (2019) 20:22
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