Targeting of Nanoparticles to the Cerebral Vasculature after Traumatic Brain Injury
Serena Omo-Lamai, Jia Nong, Walking Croup,Brian J. Kelley, Jichuan Wu, Sahily Esteves-Reyes, Liam S. Chase, Vladimir R. Muzykantov, Oscar A. Marcos-Contreras, Jean-Pierre Dollé, Douglas H. Smith, Jacob S. Brenner
Abstract
Traumatic brain injury has faced numerous challenges in drug development, primarily due to the difficulty of effectively delivering drugs to the brain. However, there is a potential solution in targeted drug delivery methods involving antibody-drug conjugates or nanocarriers conjugated with targeting antibodies. Following a TBI, the blood-brain barrier (BBB) becomes permeable, which can last for years and allow the leakage of harmful plasma proteins. Consequently, an appealing approach for TBI treatment involves using drug delivery systems that utilize targeting antibodies and nanocarriers to help restore BBB integrity. In our investigation of this strategy, we examined the efficacy of free antibodies and nanocarriers targeting a specific endothelial surface marker called vascular cell adhesion molecule-1 (VCAM-1), which is known to be upregulated during inflammation. In a mouse model of TBI utilizing central fluid percussion injury, free VCAM-1 antibody did not demonstrate superior targeting when comparing sham vs. TBI brain. However, the administration of VCAM-1-targeted nanocarriers (liposomes) exhibited a 10-fold higher targeting specificity in TBI brain than in sham control. Flow cytometry and confocal microscopy analysis confirmed that VCAM-1 liposomes were primarily taken up by brain endothelial cells post-TBI. Consequently, VCAM-1 liposomes represent a promising platform for the targeted delivery of therapeutics to the brain following traumatic brain injury.
Introduction
Traumatic brain injury (TBI) results in 230,000 hospitalizations and 50,000 deaths in the US each year annually [1]. While there have been multiple TBI clinical treatment trials, to-date none have been successful [2–9]. One major challenge is that most candidate drugs have poor accumulation in the brain, due to exclusion by the blood-brain barrier (BBB). One potential solution to this problem is to develop targeted drug delivery vehicles that can localize drugs to the injured brain. Two major platforms currently exist for targeted drug delivery. First, monoclonal antibodies that can be conjugated to small molecule drugs or siRNA, forming antibody-drug conjugates (ADCs) [10–14]. Second, nano-scale drug carriers (nanocarriers) that can be loaded with drugs and then covalently conjugated to antibodies that target a particular organ or cell type [15–17]. Both of these strategies have been investigated for general brain delivery [18,19] but with little attention to TBI specifically. Therefore, here we examined the potential of brain targeting after TBI through monoclonal antibodies and targeted nanocarriers.
Materials and methods
Materials
DPPC (dipalmitoyl phosphatidylcholine), cholesterol, and DSPE-PEG2000-azide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (ammonium salt)) were purchased from Avanti Polar Lipids (Alabaster, Alabama). All other chemicals and reagents were purchased from SigmaAldrich (St. Louis, MO), unless specifically noted.
Liposome preparation and characterization
Liposomes were formulated using the thin-film hydration method. Lipids were dissolved in chloroform and combined in a borosilicate glass tube. Chloroform was evaporated by blowing nitrogen over the solution until visibly dry (approximately15 minutes) then putting the tube under vacuum for greater than 1 hour. Dried lipid films were hydrated with phosphate buffered saline to a total lipid concentration of 20mM. The rehydrated lipid solution was vortexed and sonicated in a bath sonicator until visually homogeneous (approximately 1 minute each of vortexing and sonication). The solution was then extruded twenty-one times through a 0.2 μm polycarbonate filter. Liposomes were heated to approximately 50°C (just above the phase transition temperature of DPPC) during vortexing and extrusion. Dynamic light scattering (DLS) measurements of hydrodynamic particle size, distribution, and polydispersity index were made using a Zetasizer Pro ZS (Malvern Panalytical, Malvern UK).
Results
VCAM-1 antibody localizes significantly to the brain in sham and TBI mice
To assess the accessibility of VCAM-1 antibody in TBI brain, twenty-four hours following a sham or mild-to-moderate central fluid percussion injury to mice (Fig 1A) [37], we IV injected radiolabeled monoclonal antibody (5μg mAb per animal), or untargeted control IgG. The antibodies were allowed to circulate for 30 minutes prior to animal sacrifice (Fig 1B). VCAM-1 antibody accumulated in the brain at significantly higher levels than control IgG antibodies in both sham and TBI mice (Fig 1C and 1D, S1 Table). In sham mice, VCAM-1 antibody was taken up ~6-fold more than untargeted IgG control. By contrast, in TBI mice, VCAM-1 antibody was taken up ~2.5 fold more than IgG. It is worth noting that the IgG accumulation in the TBI brain is ~8 fold higher than that of naïve brain (black dashed line). Notably, IgG circulates well and accumulates minimally in the healthy brain. These results suggested that TBI induces capillary leakage, though at this point we cannot say if IgG extravasation into the TBI brain is due to transport that is transcellular, paracellular, or via frank hemorrhage. Additionally, the extravasated IgG in TBI brain contributed to the lower fold-improvements of VCAM-1 antibody vs. IgG control in TBI vs. sham brain.
Discussion
Acute brain injuries of various ethiologies display the common pathology of neurovascular inflammation. Upon inflammation, endothelial cells along the BBB are activated and overly express cellular adhesion molecules, including VCAM-1. In contrast to IgG, which is constantly recycled back to the plasma via the neonatal Fc receptor after cell uptake [40], IV administration of VCAM-1 antibody binds endothelium once entering the circulation. The high accessibility of BBB endothelium (surface area of ~20 m2 [41]) and upregulated expression after neurovascular inflammation in TBI makes VCAM-1 a promising target for drug delivery to the brain.
Acknowledgments
We thank Muzykantov and Brenner lab members for their technical and mental support. We also thank Hui Wei for her help with imaging.
Citation: Omo-Lamai S, Nong J, Savalia K, Kelley BJ, Wu J, Esteves-Reyes S, et al. (2024) Targeting of nanoparticles to the cerebral vasculature after traumatic brain injury. PLoS ONE 19(6): e0297451. https://doi.org/10.1371/journal.pone.0297451
Editor: Kazuhiko Kibayashi, School of Medicine, Tokyo Women’s Medical University, JAPAN
Received: June 14, 2023; Accepted: January 4, 2024; Published: June 10, 2024
Copyright: © 2024 Omo-Lamai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: S.O. received funding from the American Heart Association (Grant 23PRE1014444). J.N. received funding from the American Heart Association (Grant 916172). B.J.K received funding from National Health Institute (K08-NS110929). O.A.M.-C. received funding from the American Heart Association (Grant 19CDA34590001). J.S.B. and V.M.R. received support from the Cardiovascular Institute of the University of Pennsylvania. V.M.R. received funding from National Institute of Health (R01 HL155106, R01 HL128398, R01 HL143806). J.S.B. received funding from National Institute of Health (K08-HL-138269, R01-HL-153510, R01-HL-160694, R01-HL-157189, R21-AI-166778-01). D.H.S received funding from the Paul G. Allen Family Foundation and National Institute of Health (U54 NS115322). AHA: https://www.heart.org/en/get-involved/ways-to-give form=FUNPHPZDXBX&s_src=23L511AEMG&s_subsrc=fy23_jun_sem_google_text_&utm_mediu m=paid&utm_campaign=dr+fy23+june&utm_source=sem+google&utm_content=prospecting- https://www.med.upenn.edu/cvi/funded-dream-teams.html NIH: https://www.nih.gov/grants-funding The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0297451#abstract0
