ABSTRACT
The blood-brain barrier (BBB) is a highly restrictive barrier at the interface between the brain and the vascular system. Even under BBB dysfunction, it is extremely difficult to deliver therapies across the barrier, limiting the options for treatment of neurological injuries and disorders. To circumvent these challenges, there is interest in developing therapies that directly engage with the damaged BBB to restore its function. Previous studies revealed that poloxamer 188 (P188), a water-soluble triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), partially mitigated BBB dysfunction in vivo. In the context of stabilization of the damaged BBB, the mechanism of action of PEO-PPO block copolymers is unknown, and there has been minimal exploration of polymers beyond P188. In this study, a human-based in vitro BBB model under oxidative stress was used to investigate polymer-BBB interactions since oxidative stress is closely linked with BBB dysfunction in many neurological injuries and disorders. PEO-PPO block copolymers of varied numbers of chemically distinct blocks, PEO block length, and functionality of the end group of the PPO block were assessed for their efficacy in improving key physiological readouts associated with BBB dysfunction. While treatment with P188 did not mitigate damage in the in vitro BBB model, treatment with three diblock copolymers improved barrier integrity under oxidative stress to a similar extent. Of the considered variations in the block copolymer design, the reduction in the number of chemically distinct blocks had the strongest influence on therapeutic function. The demonstrated efficacy of three alternative PEO-PPO diblock copolymers in this work reveals the potential of these polymers as a class of therapeutics that directly treat the damaged BBB, expanding the options for treatment of neurological injuries and disorders.
ABSTRACT
Maintaining the integrity of cell membranes is indispensable for cellular viability. Poloxamer 188 (P188), a poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer with a number-average molecular weight of 8700 g/mol and containing 80% by mass PEO, protects cell membranes from various external injuries and has the potential to be used as a therapeutic agent in diverse applications. The membrane protection mechanism associated with P188 is intimately connected with how this block copolymer interacts with the lipid bilayer, the main component of a cell membrane. Here, we report the distribution of P188 in a model lipid bilayer comprising 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) using neutron reflectivity (NR) and atomic force microscopy (AFM). We also investigated the association of a PEO homopolymer (PEO8.4K; Mn = 8400 g/mol) that does not protect living cell membranes. These experiments were conducted following incubation of a 4.5 mmol/L polymer solution in a buffer that mimics physiological conditions with supported POPC bilayer membranes followed by washing with the aqueous medium. In contrast to previous reports, which dealt with P188 and PEO in salt-free solutions, both P188 and PEO8.4K penetrate into the inner portion of the lipid bilayer as revealed by NR, with approximately 30% by volume occupancy across the membrane without loss of bilayer structural integrity. These results indicate that PEO is the chemical moiety that principally drives P188 binding to bilayer membranes. No defects or phase-separated domains were observed in either P188- or PEO8.4K-incubated lipid bilayers when examined by AFM, indicating that polymer chains mingle homogeneously with lipid molecules in the bilayer. Remarkably, the breakthrough force required for penetration of the AFM tip through the bilayer membrane is unaffected by the presence of the large amount of P188 and PEO8.4K.
Subject(s)
Lipid Bilayers , Propylene Glycols , Polyethylene Glycols , PolymersABSTRACT
BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene. 40% of X-ALD patients will convert to the deadly childhood cerebral form (ccALD) characterized by increased permeability of the brain endothelium that constitutes the blood-brain barrier (BBB). Mutation information and molecular markers investigated to date are not predictive of conversion. Prior reports have focused on toxic metabolic byproducts and reactive oxygen species as instigators of cerebral inflammation and subsequent immune cell invasion leading to BBB breakdown. This study focuses on the BBB itself and evaluates differences in brain endothelium integrity using cells from ccALD patients and wild-type (WT) controls. METHODS: The blood-brain barrier of ccALD patients and WT controls was modeled using directed differentiation of induced pluripotent stem cells (iPSCs) into induced brain microvascular endothelial cells (iBMECs). Immunocytochemistry and PCR confirmed characteristic expression of brain microvascular endothelial cell (BMEC) markers. Barrier properties of iBMECs were measured via trans-endothelial electrical resistance (TEER), sodium fluorescein permeability, and frayed junction analysis. Electron microscopy and RNA-seq were used to further characterize disease-specific differences. Oil-Red-O staining was used to quantify differences in lipid accumulation. To evaluate whether treatment with block copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO-PPO) could mitigate defective properties, ccALD-iBMECs were treated with PEO-PPO block copolymers and their barrier properties and lipid accumulation levels were quantified. RESULTS: iBMECs from patients with ccALD had significantly decreased TEER (2592 ± 110 Ω cm2) compared to WT controls (5001 ± 172 Ω cm2). They also accumulated lipid droplets to a greater extent than WT-iBMECs. Upon treatment with a PEO-PPO diblock copolymer during the differentiation process, an increase in TEER and a reduction in lipid accumulation were observed for the polymer treated ccALD-iBMECs compared to untreated controls. CONCLUSIONS: The finding that BBB integrity is decreased in ccALD and can be rescued with block copolymers opens the door for the discovery of BBB-specific molecular markers that can indicate the onset of ccALD and has therapeutic implications for preventing the conversion to ccALD.