Purpose: Ischemic stroke can result in dysfunction and death of brain neurons. It has been reported that brain-derived neurotrophic factor (BDNF) is an essential nervous system growth factor in the brain which maintains physiological processes of the normal, intact adult brain. Insufficient delivery of BDNF is a major challenge that limits its potential to treat stroke, even during a stroke injury where the blood-brain barrier (BBB) might have been transiently disrupted. Our goal is to deliver BDNF DNA using self-assembled polymer-based nanoparticles (NPs) to the BBB. DNA NPs are formed by electrostatic interaction between amine groups of PEG-DET and phosphate groups of DNA. We used a triblock amphiphilic copolymer, Pluronic P84 (P84) as a safe excipient to increase the transfection efficiency of PEG-DET* DNA NPs. Our specific goal here is to confirm the presence of functional tight junctions (TJ) in a human cerebral microvascular endothelial cell line (hCMEC/D3, a cell model of the human BBB). We further wanted to determine if DNA NP treatment adversely affects TJ functionality. Results of the above studies will be presented.
* PEG-DET: Poly (ethylene glycol)5k-poly (L-aspartate-diethylenetriamine)48
Methods: 1. DNA NP Transfection assay: The hCMEC/D3 cells were cultured until 100% confluency and transfected with PEG-DET DNA (+ P84) NPs for 4 hrs and luciferase (model gene) expression was measured 24 h post-transfection.
2. Luminescent ATP assay: The hCMEC/D3 cells were cultured until 100% confluency. After four-hour transfection, the transfection mixture was removed and a CellTiter-Glo® assay was used to evaluate cell viability.
3. Lucifer yellow (LY) Permeability assay: hCMEC/D3 monolayers were cultured on permeable Transwell insert supports and LY was used as a marker of paracellular permeability.
Results: DNA NP/P84 transfected hCMEC/D3 cells showed a 13 to 21-fold increase in gene expression compared to DNA NP alone (no P84) treated cells without any cellular toxicity. The trend of LY apparent permeability (Papp) values was similar to the changes in transendothelial electrical resistance values in cultured hCMEC/D3 cells, as reported earlier by Eigenmann et al . A small (although not significant) increase in LY Papp was noted in the DNA NP/P84-transfected cells compared to control, untreated cells.
 Eigenmann , G.X., Kwang S Kim, Ashlee V Moses, Matthias Hamburger and Mouhssin Oufir*, Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood–brain barrier model for drug permeability studies. Fluids and Barriers of the CNS, 2013. 10(33).
Conclusion: DNA NP with P84 is a safe formulation to transfect brain endothelial cells. PEG-DET DNA NPs with P84 had no significant effect on tight junction functionality and barrier integrity.