Targeting of nanoparticles to the cerebral vasculature after traumatic brain injury

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.

Fig 1. In TBI mice, VCAM-1 antibody has significantly higher brain uptake than untargeted IgG control.

(A) To prepare mice for the central fluid percussion injury, a sterile Leur-lock syringe hub was first attached to the craniotomy site which was then filled with saline. Using a fluid percussion injury device, a mild-to-moderate injury was administered by releasing the pendulum onto a fluid-filled piston in order to exert fluid pressure upon the dura. (B) Timeline of biodistribution, flow cytometry and histology experiments of monoclonal antibodies or targeted-nanocarriers against VCAM-1 or untargeted IgG control. Comparing the brain uptake of VCAM-1 to IgG in sham (C) and TBI (D) shows that antibodies against endothelial targets accumulate in the brain significantly more than control IgG antibodies (%ID/g: % of injected dose per gram of tissue). Notably, there is no significant difference between the brain uptake in sham and TBI mice. Dashed line represents naïve level of IgG (black) vs. VCAM-1 (red). (E) Similarly, no significant differences were observed in the biodistribution of VCAM-1 and IgG antibodies in other organs between sham and TBI mice. N≥3 and data shown represents mean ± SEM; Comparisons were made by student’s t-test. *p<0.05, ***p<0.001.

Fig 2. VCAM-1-targeted liposomes achieve the highest brain uptake in TBI mice and accumulate primarily with endothelial cells.

(A) In sham mice, there is no significant difference in brain uptake between VCAM-1 and IgG liposomes, but in TBI mice, VCAM-1 liposomes are taken up in the brain significantly more than IgG liposomes. (B) No significant differences were observed in the biodistribution of VCAM-1 and IgG liposomes in other organs between sham and TBI mice. (C) Immunospecificity index shows that VCAM-1 antibody has lower targeting specificity in TBI brain. In contract, VCAM-1-targeted liposomes exhibited 10-fold higher targeting specificity to TBI brain, compared to sham. (D) Several cellular populations were recovered during brain flow cytometry: endothelial cells (CD31-high, CD45-neg), leukocytes (CD45-high) and microglia (CD45-mid), and a significantly higher proportion of leukocytes were recovered compared to endothelial cells. Figure calculated as total of 100%. (E) Among all the nanocarrier-positive cells, VCAM-1-liposomes primarily associated with endothelial cells. (F) Within specific cell type, endothelial cells represented a significantly higher proportion of VCAM-1-liposome-positive cells than leukocytes. (G) Using confocal microscopy, we demonstrate the delivery of VCAM-1-targeted liposomes to the endothelium (CD31+) of TBI mouse brains. Scale bar = 100 μm. N≥3 and data shown represents mean ± SEM; For (E), statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test. For all other figures, comparisons were made by 1-way ANOVA with Dunnett’s post hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.