Comparison of the Anti-inflammatory Activity and Cellular Interaction of Brush Polymer–N-Acetyl Cysteine Conjugates in Human and Murine Microglial Cell Lines

Abstract

Microglia-mediated neuroinflammation is commonly associated with neurodegeneration and has been implicated in several neurological disorders, such as Alzheimer's disease and Parkinson's disease. Therefore, it is crucial to develop a detailed understanding of the interaction of potential nanocarriers with microglial cells to efficiently deliver anti-inflammatory molecules. In this study, we applied brush polymers as a modular platform to systematically investigate their association with murine (BV-2) and human (HMC3) microglial cell lines in the presence and absence of the pro-inflammatory inducer lipopolysaccharide (LPS) using flow cytometry. Brush polymers of different sizes and shapes, ranging from ellipsoid to worm-like cylinders, were prepared through a combination of the two building blocks carboxylated Nacylated poly(aminoester)s (NPAEs)-based polymers and poly(2-ethyl-2oxazoline)-NH 2 (PEtOx-NH 2 ) and characterized by 1 H NMR spectroscopy, size exclusion chromatography, and small-angle neutron scattering. Generally, ellipsoidal particles showed the highest cellular association. Moreover, while no significant differences in murine cell association were observed, the brush polymers revealed a significant accumulation in LPS-activated human microglia compared to resting cells, emphasizing their higher affinity to activated HMC3 cells. Brush polymers with the highest cell association were further modified with the anti-inflammatory agent N-acetyl cysteine (NAC) in a reversible manner. The brush polymer−NAC conjugates were found to significantly attenuate the production of interleukin 6 (p < 0.001) in LPS-activated HMC3 cells compared to LPS-activated BV-2 cells. Thus, the presented brush polymer− NAC conjugates showed a high anti-inflammatory activity in human microglia, suggesting their potential for disease-targeted therapy of microglial-mediated neuroinflammation in the future.

Folic Acid-Conjugated Brush Polymers Show Enhanced Blood–Brain Barrier Crossing in Static and Dynamic In Vitro Models Toward Brain Cancer Targeting Therapy

“…According to numerous studies, nanoparticles with higher aspect ratios have shown increased interaction with cell membranes. This is often attributed to their higher degree of accessible surface area, which provides more binding or association sites for interaction with cell membranes. − In contrast, as previously observed with microglial cells, polymeric bottlebrush of ellipsoid shape with a length of 5.7 nm (M50-PEtOx 10 ) shows the highest interaction with hCMEC/D3 cells compared to elliptical worm-like chain polymers of 20.8 nm (M400-PEtOx 10 ) and 35.4 nm (M1000-PEtOx 10 or M1000-PEtOx 35 ) length. Based on previous studies, the wrapping time for elongated bottlebrush-like nanoparticles within the cells is longer than for ellipsoid/spherical nanoparticles due to their higher surface area.…”

“…17 As an initial assessment, we investigated the impact of the physical parameters of our polymeric brushes, including size and shape, on their brain endothelial cell association. A library of nonfunctionalized polymeric brushes with different sizes and shapes (M50-PEtOx 10 , M400-PEtOx 10 , M1000-PEtOx 10 , and M1000-PEtOx 35 ) prepared previously 16 was further exploited to understand the role of morphology on the interaction with brain endothelial cells before incorporation of the targeting ligand.…”

“…In this study, a polymeric nanoplatform based on NPAEs and PEtOx for brain drug delivery is presented. A library of bottlebrush-like polymeric systems with different sizes/shapes prepared in a previous study (Table S1) 16 was first investigated for their interaction with human brain endothelial cells (hCMEC/D3) in order to select the most promising candidates to conjugate to FA. To enable FA conjugation, heterotelechelic PEtOx with α-amine and ω-N 3 was synthesized via phthalimide amine initiation and sodium azide (NaN 3 ) termination, resulting in NH 2 -PEtOx-N 3 after phthalimide deprotection and effectively incorporated into comb polymer by a single step of amidation.…”

“…46 M50 was prepared based on a previous literature protocol. 16 Instrumentation. Microwave polymerization was carried out in a CEM Discover SP microwave synthesizer.…”

“…Polymeric nanoparticles have been proposed as one promising modality for brain drug delivery due to their advantages, including enhanced solubility of drug payload, reduced drug metabolism, biodegradability, enhanced therapeutic efficacy, and reduced toxicity relative to the administration of free drugs. , Additionally, polymeric nanoparticles can be easily engineered by tuning their physicochemical properties based on the targeted application with enhanced delivery to brain. − Nevertheless, these studies are still far from optimal as the accumulation in the liver reduces the overall availability of the drug in the CNS. , One of the common ways to enhance drug delivery across the BBB is to adorn the surface of these bespoke nanomaterials with targeting ligands that can be recognized by the receptors expressed on the BBB and internalized via the process of endocytosis. ,− In addition to the nanoparticle size, shape, surface charge, − ligand type, conjugation strategy, orientation, and ligand density have recently emerged as critical parameters that significantly dictate the mechanism and kinetics of its cellular interaction. , In a recent review article, it was demonstrated that when the ligand density increases, the cellular association increases to a limit, after which a constant or decreased association is observed . Interestingly, targeted nanoparticles often qualitatively report the ligand density as ‘low’, ‘intermediate’, and ‘high’, or as a percentage and only a few reports quantitatively determine the number of ligands per nanoparticles.…”