Targeted Nanocarriers Co-Opting Pulmonary Intravascular Leukocytes for Drug Delivery to the Injured Brain

Abstract

Ex vivo-loaded white blood cells (WBC) can transfer cargo to pathological foci in the central nervous system (CNS). Here we tested affinity ligand driven in vivo loading of WBC in order to bypass the need for ex vivo WBC manipulation. We used a mouse model of acute brain inflammation caused by local injection of tumor necrosis factor alpha (TNF-α). We intravenously injected nanoparticles targeted to intercellular adhesion molecule 1 (anti-ICAM/NP). We found that (A) at 2 h, >20% of anti-ICAM/NP were localized to the lungs; (B) of the anti-ICAM/NP in the lungs >90% were associated with leukocytes; (C) at 6 and 22 h, anti-ICAM/NP pulmonary uptake decreased; (D) anti-ICAM/NP uptake in brain increased up to 5-fold in this time interval, concomitantly with migration of WBCs into the injured brain. Intravital microscopy confirmed transport of anti-ICAM/NP beyond the blood-brain barrier and flow cytometry demonstrated complete association of NP with WBC in the brain (98%). Dexamethasone-loaded anti-ICAM/liposomes abrogated brain edema in this model and promoted anti-inflammatory M2 polarization of macrophages in the brain. In vivo targeted loading of WBC in the intravascular pool may provide advantages of coopting WBC predisposed to natural rapid mobilization from the lungs to the brain, connected directly via conduit vessels.

Keywords: brain; drug delivery; inflammation; nanoparticles; pharmacokinetics; white blood cells.

Figure 1

Local injection of TNF-α into the brain induces a systemic response. Following IV injection of αCD45, (A) blood, (D) lung, and (I) brain targeting was assessed at several time points post-TNF. Similar studies were performed for αICAM biodistribution in (B) blood, (E) lungs, and (J) brain. Data represented as percent of injected dose per gram tissue (%ID/g). Mice were injected with 5 μg mAb at the designated time points following TNF-α injection and mAb was allowed to circulate for 30 min prior to organ harvest. Naïve mice are represented as t = 0 h. Flow cytometry was used to measure the dynamics of ICAM⁺ leukocytes in the (C) peripheral blood 2 h after TNF-α injection, (G,H) pulmonary intravascular compartment 2 h after TNF-α injection, and (K) brain 24 h after TNF-α injection. (F) Histological analysis of lungs 2 h after TNF-α injection into the brain. Red: Leukocytes (CD45); Green: Autofluorescence (tissue structure); Scale bar = 50 μm. N = 3 mice/group. Comparisons in A, B, D, E, G, and H made via one-way ANOVA with Dunnett’s posthoc test vs naïve and in C, F, and I via unpaired Student’s t test.

Figure 2

αICAM and αCD45 mAbs rapidly accumulate in the lungs and then migrate to the brain. (A) Schematic of proposed mechanism underlying leukocyte migration. (B) PK study timeline. Lung and brain pharmacokinetics of mAbs directed against (C) PECAM, (D) ICAM, and (E) CD45 following IV injection 2 h post-TNF-α injury. Uptake of mAbs directed against (F) PECAM, (G) ICAM, and (H) CD45 in clearance organs (liver, spleen) following IV injection 2 h post-TNF-α injury. Time points reflect the time post-mAb injection when organs were harvested. Solid bars: 30 min, Striped bars: 4 h, Checkered bars: 22 h. Data represented as mean ± SEM. Comparisons made by one-way ANOVA with Tukey’s posthoc test. N = 3 mice/group. Portions of figure created using www.biorender.com.

Figure 3

ICAM-targeted nanoparticles accumulate in the inflamed brain. (A) Study timeline. Pharmacokinetics of (B) polystyrene nanoparticles, (C) liposomes, and (D) lipid nanoparticles in the lungs and brain following injection. Kinetic changes in the ratio of nanoparticles in brain vs lungs for (E) polystyrene nanoparticles, (F) liposomes, and (G) lipid nanoparticles. Pharmacokinetics in clearance organs of (H) polystyrene nanoparticles, (I) liposomes, and (J) lipid nanoparticles. (K) Transmission electron microscopy of ICAM-targeted polystyrene nanoparticles in lung endothelium and leukocytes 30 min postinjection. (L) Cranial window intravital microscopy of ICAM-targeted polystyrene nanoparticles in the TNF-α injured brain. Data represented as mean ± SEM. Comparisons were made by one-way ANOVA with Tukey’s posthoc test. N ≥ 3 mice/group.

Figure 4

Cellular specificity of ICAM-targeted polystyrene nanoparticles. (A) Flow cytometry was performed on single cell suspensions obtained from lungs and brain at the designated times post-nanoparticle injection. (B) Relative affinity of nanoparticles for endothelial cells and leukocytes was assessed by considering the fraction of cells that took up nanoparticles. The fraction of nanoparticles recovered in (C) lungs and (D) brain that were associated with specific cell types. Leukocytes: CD45⁺, Endothelium: CD31⁺CD45^–. Histology of brain tissue sections collected 22 h postinjection of polystyrene nanoparticles in TNF-α challenged mice. Nanoparticle association with macrophages (CD68⁺) and endothelial cells (VCAM⁺) was measured for (E,F) ICAM-targeted and (G,H) IgG nanoparticles. Scale bar: 50 μm. Data represented as mean ± SEM. N = 3/group.

Figure 5

ICAM-targeted dexamethasone (Dex) liposomes protect mice from TNF-induced brain edema. (A) Experimental timeline. (B) Protective effects of ICAM-targeted dexamethasone liposomes (0.5 mg/kg dexamethasone). As controls for Dex-loaded liposomes, equivalent doses of empty IgG or ICAM-targeted liposomes were tested. % protection was calculated assuming 100% protection as equivalent to edema induced by sham injury and 0% protection as equivalent to edema induced by TNF injury without treatment (Supplemental Figure 19). (C) Impact of treatment strategies on leukocyte abundance and phenotype in the brain as measured by flow cytometry. Data displayed as mean ± SEM. Comparisons made by one-way ANOVA with Dunnett’s posthoc test vs untreated (solid line, 0% protection) in B and Tukey’s posthoc test in C. N ≥ 3/group.