Biotransformation modulates the penetration of metallic nanomaterials across an artificial blood–brain barrier model

Understanding the potential of nanomaterials (NMs) to cross the blood–brain barrier (BBB), as a function of their physicochemical properties and subsequent behavior, fate, and adverse effect beyond that point, is vital for evaluating the neurological effects arising from their unintentional entry into the brain, which is yet to be fully explored. This is not only due to the complex nature of the brain but also the existing analytical limitations for characterization and quantification of NMs in the complex brain environment. By using a fit-for-purpose analytical workflow and an in vitro BBB model, we show that the physiochemical properties of metallic NMs influence their biotransformation in biological matrices, which in turn modulates the transport form, efficiency, amounts, and pathways of NMs through the BBB and, consequently, their neurotoxicity. The data presented here will support in silico modeling and prediction of the neurotoxicity of NMs and facilitate the tailored design of safe NMs.

Is LRP2 Involved in Leptin Transport over the Blood-Brain Barrier and Development of Obesity?

The mechanisms underlying the transport of leptin into the brain are still largely unclear. While the leptin receptor has been implicated in the transport process, recent evidence has suggested an additional role of LRP2 (megalin). To evaluate the function of LRP2 for leptin transport across the blood-brain barrier (BBB), we developed a novel leptin-luciferase fusion protein (pLG), which stimulated leptin signaling and was transported in an in vitro BBB model based on porcine endothelial cells. The LRP inhibitor RAP did not affect leptin transport, arguing against a role of LRP2. In line with this, the selective deletion of LRP2 in brain endothelial cells and epithelial cells of the choroid plexus did not influence bodyweight, body composition, food intake, or energy expenditure of mice. These findings suggest that LRP2 at the BBB is not involved in the transport of leptin into the brain, nor in the development of obesity as has previously been described.

Amylin-mediated regulation of LRP1 by miR-103/107 induces cerebral microvascular dysfunction and impairs β-amyloid efflux

Background: The cerebral small blood vessels of individuals with Alzheimer’s Disease (AD) often have deposits of amylin, an amyloid-forming protein secreted in the blood by pancreatic β-cells. To determine whether systemic pancreatic amylin dyshomeostasis impairs amyloid β (Aβ) efflux across the blood-brain barrier (BBB), we studied cerebral microvessels in humans and rats with pancreatic expression of amyloidogenic human amylin, and evaluated the effect of human amylin in an in vitro BBB model. Methods: Brain sections from AD and cognitively unimpaired individuals were co-stained with anti-Aβ and anti-amylin antibodies. In vivo analyses of Aβ efflux across the BBB were carried out in aged rats that express amyloid-forming human amylin in pancreatic β-cells and littermates expressing non-amyloidogenic rat amylin. We also used an in vitro BBB model of Aβ transcytosis in which the endothelial cell monolayer was exposed to amylin-mediated stress to determine whether amylin stress downregulates LRP1, the Aβ efflux transporter. This allowed us to use pharmacology to rescue the endothelial LRP1. Results: In human AD brains, Aβ accumulation within the perivascular space frequently co-localized with deposits of amylin in the vessel wall. In rats with pancreatic expression of amyloid-forming human amylin, the high blood levels of human amylin promoted amylin deposition in brain capillaries, increased brain Aβ level, lowered plasma-to-brain Aβ ratio and suppressed expression of LRP1 protein. In vitro BBB model experiment revealed that amylin-induced stress downregulates LRP1 in endothelial cells through a miRNA-based translational repression mechanism. Conclusions: High blood human amylin levels cause cerebral microvascular dysfunction and interfere with Aβ efflux across the BBB through miRNA-mediated LRP1 downregulation. Lowering the blood amylin level in early AD could improve Aβ clearance from the brain.

An Elvitegravir Nanoformulation Crosses the Blood–Brain Barrier and Suppresses HIV-1 Replication in Microglia

Even with an efficient combination of antiretroviral therapy (ART), which significantly decreases viral load in human immunodeficiency virus type 1 (HIV-1)-positive individuals, the occurrence of HIV-1-associated neurocognitive disorders (HAND) still exists. Microglia have been shown to have a significant role in HIV-1 replication in the brain and in subsequent HAND pathogenesis. However, due to the limited ability of ART drugs to cross the blood–brain barrier (BBB) after systemic administration, in addition to efflux transporter expression on microglia, the efficacy of ART drugs for viral suppression in microglia is suboptimal. Previously, we developed novel poly (lactic-co-glycolic acid) (PLGA)-based elvitegravir nanoparticles (PLGA-EVG NPs), which showed improved BBB penetration in vitro and improved viral suppression in HIV-1-infected primary macrophages, after crossing an in vitro BBB model. Our objective in the current study was to evaluate the efficacy of our PLGA-EVG NPs in an important central nervous system (CNS) HIV-1 reservoir, i.e., microglia. In this study, we evaluated the cyto-compatibility of the PLGA-EVG NPs in microglia, using an XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay and cellular morphology observation. We also studied the endocytosis pathway and the subcellular localization of PLGA NPs in microglia, using various endocytosis inhibitors and subcellular localization markers. We determined the ability of PLGA-EVG NPs to suppress HIV-1 replication in microglia, after crossing an in vitro BBB model. We also studied the drug levels in mouse plasma and brain tissue, using immunodeficient NOD scid gamma (NSG) mice, and performed a pilot study, to evaluate the efficacy of PLGA-EVG NPs on viral suppression in the CNS, using an HIV-1 encephalitic (HIVE) mouse model. From our results, the PLGA-EVG NPs showed ~100% biocompatibility with microglia, as compared to control cells. The internalization of PLGA NPs in microglia occurred through caveolae-/clathrin-mediated endocytosis. PLGA NPs can also escape from endo-lysosomal compartments and deliver the therapeutics to cells efficiently. More importantly, the PLGA-EVG NPs were able to show ~25% more viral suppression in HIV-1-infected human-monocyte-derived microglia-like cells after crossing the in vitro BBB compared to the EVG native drug, without altering BBB integrity. PLGA-EVG NPs also showed a ~two-fold higher level in mouse brain and a trend of decreasing CNS HIV-1 viral load in HIV-1-infected mice. Overall, these results help us to create a safe and efficient drug delivery method to target HIV-1 reservoirs in the CNS, for potential clinical use.

VLA-4 mediated adhesion of melanoma cells on the blood–brain barrier is the critical cue for melanoma cell intercalation and barrier disruption

Melanoma is the most aggressive skin cancer in humans. One severe complication is the formation of brain metastasis, which requires extravasation of melanoma cells across the tight blood–brain barrier (BBB). Previously, VLA-4 has been assigned a role for the adhesive interaction of melanoma cells with non-BBB endothelial cells. However, the role of melanoma VLA-4 for breaching the BBB remained unknown. In this study, we used a mouse in vitro BBB model and imaged the shear resistant arrest of melanoma cells on the BBB. Similar to effector T cells, inflammatory conditions of the BBB increased the arrest of melanoma cells followed by a unique post-arrest behavior lacking immediate crawling. However, over time, melanoma cells intercalated into the BBB and compromised its barrier properties. Most importantly, antibody ablation of VLA-4 abrogated melanoma shear resistant arrest on and intercalation into the BBB and protected the BBB from barrier breakdown. A tissue microarray established from human brain metastasis revealed that indeed a majority of 92% of all human melanoma brain metastases stained VLA-4 positive. We propose VLA-4 as a target for the inhibition of brain metastasis formation in the context of personalized medicine identifying metastasizing VLA-4 positive melanoma.