(M0930-03-18) Exofection as a Therapeutic Modality: Restoring P-gp Activity via Trophoblast-Derived Extracellular Vesicles in Neuroinflammatory Disorders

Purpose: P-glycoprotein (P-gp/ABCB1), an ATP-dependent efflux transporter, maintains barrier integrity by clearing xenobiotics and toxic metabolites¹. Downregulation of P-gp is associated with impaired amyloid-β (Aβ) clearance and neuroinflammation in Alzheimer’s disease (AD)². Our previous findings identified that trophoblast-derived extracellular vesicles (CTC-EVs) can restore P-gp function at the feto-maternal interface through exofection. This study investigated whether CTC-EV-mediated exofection could restore P-gp activity in brain endothelial cells (BECs) and mitigate neurodegenerative pathology in AD.

Methods: CTC-derived EVs were isolated and characterized by nanoparticle tracking analysis and western blotting for P-gp and exosomal markers. BECs were exposed to inflammatory stimuli (LPS 500 ng/mL, 48 h) and treated with CTC-EVs. P-gp expression was assessed by immunofluorescence and quantified using mean fluorescence intensity (MFI) analysis, with statistical comparisons to controls. Functional efflux activity was evaluated using Calcein-AM dye accumulation assays. In vivo, 3xTg-AD mouse models of AD and related late-onset dementia (AD/ADRD) were treated with CTC-EVs intravenously at dose of 1E+09 EVs for five days. P-gp expression in the frontal cortex and hippocampus was analyzed by Western blot and immunohistochemistry. Drug clearance kinetics were evaluated in P-gp knockout mice treated with CTC-EVs to validate transporter recovery.

Results: Inflammatory conditions significantly reduced P-gp expression in BECs (MFI reduced by 41.3%, p = 0.0084), while CTC-EV treatment rescued the loss of P-gp expression (increase of 46.7% over LPS alone, p = 0.0121). Relative intensity measurements from western blots confirmed these findings, with P-gp levels increasing 2.1-fold post-treatment. Calcein-AM assays revealed enhanced efflux activity, with a significant reduction in intracellular fluorescence following EV treatment (p < 0.001). In vivo, 3xTg-AD treated with CTC-EVs showed increased P-gp expression in both frontal cortex (38.6% increase, p = 0.0143) and hippocampus (42.1%, p = 0.0117), alongside reduced Aβ burden and markers of neuroinflammation. Pharmacokinetic studies in P-gp KO mice showed partial restoration of drug clearance in the brain post-EV treatment, further supporting functional exofection.

Conclusion: CTC-derived EVs facilitate targeted delivery of functional P-gp to deficient endothelial cells and restore transporter activity under inflammatory and neurodegenerative conditions. These findings provide preclinical evidence for EV-mediated exofection as a promising therapeutic platform to combat P-gp downregulation in AD/ADRD.

References: 1. Huang L, Chen Y, Liu R, Li B, Fei X, Li X, Liu G, Li Y, Xu B, Fang W. P-Glycoprotein Aggravates Blood Brain Barrier Dysfunction in Experimental Ischemic Stroke by Inhibiting Endothelial Autophagy. Aging Dis. 2022 Oct 1;13(5):1546-1561. doi: 10.14336/AD.2022.0225. PMID: 36186136; PMCID: PMC9466967.
2. Park R, Kook SY, Park JC, Mook-Jung I. Aβ1-42 reduces P-glycoprotein in the blood-brain barrier through RAGE-NF-κB signaling. Cell Death Dis. 2014 Jun 26;5(6):e1299. doi: 10.1038/cddis.2014.258. PMID: 24967961; PMCID: PMC4611731.

Acknowledgements: This research was supported by the National Institutes of Health (NIH), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) under award number R01HD113193 to AK.


Figure 1. Exosomal treatment restores P-glycoprotein (P-gp) expression in LPS-treated brain endothelial cells (BECs). BECs were treated with lipopolysaccharide (LPS, 500 ng/mL) for 48 hours to induce inflammation. To assess the ability of chorion trophoblast cell-derived extracellular vesicles (CTC EVs; 1 × 10⁹ particles) to restore transporter expression, BECs were co-treated with LPS and CTC EVs. (A) Immunofluorescence images (left panel) show DAPI (nuclei, blue) and P-gp (green) staining across the experimental groups: Control, LPS alone, CTC EVs alone, and LPS + CTC EVs. LPS treatment reduced P-gp expression, which was restored upon co-treatment with CTC EVs. (B) Quantification of immunofluorescence intensity (top right bar graph) confirms significant recovery of P-gp expression in the LPS + CTC EV group. (C) Western blot analysis (middle right) further validates this trend, (D) with the corresponding densitometry shown in the bottom right graph . β-actin was used as a loading control. Data represent mean ± SEM; n = 3 independent experiments.