Abstract
Brain derived neurotrophic factor (BDNF) can induce neural differentiation in stem cells and has the potential for repair of the nervous system. In this study, a polysorbate 80-coated polybutylcyanoacrylate nanocarrier (PS80 PBCA NC) was constructed to deliver plasmid DNAs (pDNAs) containing BDNF gene attached to a hypoxia-responsive element (HRE-cmvBDNF). The hypoxia-sensing mechanism of BDNF expression and inductiveness of the nano-formulation on mouse induced pluripotent stem cells (iPSCs) to differentiate into neurons following hypoxia was tested in vitro with immunofluorescent staining and Western blotting. The HRE-cmvBDNF appeared to adsorb onto the surface of PS80 PBCA NC, with a resultant mean diameter of 92.6 ± 1.0 nm and zeta potential of −14.1 ± 1.1 mV. HIF-1α level in iPSCs was significantly higher in hypoxia, which resulted in a 51% greater BDNF expression when transfected with PS80 PBCA NC/HRE-cmvBDNF than those without hypoxia. TrkB and phospho-Akt were also elevated which correlated with neural differentiation. The findings suggest that PS80 PBCA NC too can be endocytosed to serve as an efficient vector for genes coupled to the HRE in hypoxia-sensitive cells, and activation of the PI3/Akt pathway in iPSCs by BDNF is capable of neural lineage specification.
Highlights
Brain-derived neurotrophic factor (BDNF) belongs to the neutrophin family of growth factors that plays a crucial role in the survival, differentiation and synaptic plasticity of neurons in the mammalian nervous system [1]
The release of BDNF into the extracellular space may be triggered by various patterns of neural electrical activity as well as neurotransmitters and related substances [4], and the effects are mediated via activation of the tropomysin-related kinase B (TrkB) receptor-signaling pathways such as the Ras/extracellular signal regulated kinase (ERK), phosphatidylinositol-3-OH kinase (PI3K)/Akt kinase and phospholipase C-γ 1 (PLC-γ 1) pathways [5]
Various moieties have been conjugated to the BDNF for added functionality: for example, the intraperitoneal administration of the fusion-peptide in rodent models of Alzheimer’s disease that consisted of BDNF and an human immunodeficiency virus (HIV)-encoded transactivator of transcription cell-penetrating peptide designed for greater blood–brain barrier (BBB) penetration led to a significant spatial memory improvement [12]; and, BDNF conjugated with 2000DA polyethylene glycol and OX26 monoclonal antibody to the transferrin receptor was able to reduce systemic BDNF clearance by 67%, and target the BBB to decrease the infarct volume by up to 65% when it is given intravenously to rats subjected to middle cerebral artery occlusion [13,14]
Summary
Brain-derived neurotrophic factor (BDNF) belongs to the neutrophin family of growth factors that plays a crucial role in the survival, differentiation and synaptic plasticity of neurons in the mammalian nervous system [1]. The observed change in surface texture suggests adsorption of the pDNAs onto the NC, and the interactive forces are presumed to be non-covalent in nature with desorption as the main release mechanism This mode of delivery has been shown to partially protect pDNAs from nuclease degradation by electrophoretic mobility study [60], desorption or unloading of the pDNA for transcription may be better facilitated by the weak interaction between pDNA and anionic PS80 PBCA NC, the immunogenicity to these surface-bound pDNAs will need to be investigated further. Our result showed that the enhanced expression of BDNF coupled the recruitment of TrkB receptors, and activation of the PI3K/Akt signaling pathway might be responsible for the neural differentiation of iPSCs. The elevated level of HIF-1α in iPSCs under hypoxia signified an intact hypoxia-sensing mechanism, and that hypoxia could augment BDNF expression from the delivered HRE-cmvBDNF gene. We have demonstrated in this study the feasibility and neural differentiation capability of iPSCs treated by a hypoxia-sensing non-viral vector comprised of PS80 PBCA NC and HRE-cmvBDNF. The nano-formulation may be best suited for targeted BDNF-induced neuroregeneration or neuroprotection where perturbation in CNS oxygenation lies such as those seen in ischemic or hemorrhagic strokes, the modes of administration, pharmacokinetics and toxicity profile will need to be evaluated in future in vivo studies, and, the treated iPSCs may be transplanted as a cell-based BDNF delivery strategy [72,73], where the overexpressed BDNF may act on the donor stem cells, and promote neural differentiation of resident stem cells
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