Selective targeting of nanomedicine to inflamed cerebral vasculature to enhance the blood-brain barrier

Abstract

Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies.

Keywords: VCAM-1; blood–brain barrier; cerebrovascular drug targeting; drug delivery to brain; mRNA therapy.

Fig. 1.

Biodistribution of radiolabeled antibodies and immunoliposomes in naïve and TNFα-injured mice. (A, Left) Anti–ICAM-1 mAb demonstrates specific uptake in the lung (***P < 0.001 vs. IgG and VCAM-1), with a slight—but statistically significant—increase in animals receiving intrastriatal TNFα (^#P < 0.05 vs. naïve). Anti–VCAM-1 mAb, in contrast, accumulates in the brain (***P < 0.001 vs. IgG and ICAM-1) and demonstrates a >10-fold increase in both brain uptake (A, Middle) and brain:blood ratio (A, Right) following intrastriatal TNFα (***P < 0.001 vs. naïve). (B) ICAM-1 and VCAM-1 targeted immunoliposomes show nearly identical patterns of lung and brain biodistribution as their counterpart mAbs. In particular, anti–VCAM-1 liposomes demonstrate a similar ∼10-fold increase in brain uptake (B, Middle) and brain:blood ratio (B, Right) in TNFα-injured mice (***P < 0.001 vs. naïve). In all experiments, organ biodistribution was measured 30 min after i.v. injection (0.2 mg/kg of antibody and 20 mg/kg of liposome) of radiolabeled materials. mAb or immunoliposomes were given 16 h after intrastriatal TNFα injection. Each data point represents n = 3 animals with mean ± SD shown, and two-way ANOVA with Dunnett’s post hoc test was applied. Note that IgG and IgG/liposome bars are practically invisible in all panels (the data are shown in SI Appendix, Table S1). *P < 0.05 vs. IgG; **P < 0.01 vs. IgG.

Fig. 2.

SPECT imaging of immunoliposomes. Three-dimensional reconstructions (A) and average intensity projections (B) of SPECT (red) and CT (gray) signals for intrastriatal TNFα-injured mice receiving IgG or anti–VCAM-1 functionalized liposomes (20 mg/kg) bearing ¹¹¹In-DTPA (n = 3 per condition). Average intensity projections (B) encompassed SPECT and CT signal in the mouse cranium. (C) After Renyi filtering for noise removal, raw SPECT signal intensity was determined for a manually drawn field of view encompassing the cranial skull. Mean ± SD (n = 3 per group); **P < 0.01, t test. (D) Autoradiography images generated by anti–VCAM-1 functionalized liposomes bearing ¹¹¹In-DTPA in TNFα-injured brain sections. Arrows indicate the injected hemisphere, dashed lines delineate the brain hemispheres, and asterisk indicates injection site.

Fig. 3.

Intravital imaging of cerebrovascular immunoliposome distribution. Intravital microscopy was performed through a cranial window and used to demonstrate real-time localization of fluorescent VCAM-1 targeted (bottom two rows, two independent animals) vs. IgG control (top two rows, two independent animals) liposomes (green; 20 mg/kg). Merged images also show circulating leukocytes (red) labeled via i.v. injection of rhodamine dye. Left-hand panels show baseline images and accumulation of liposomes 10 min, 2 h, and 4 h after administration in naïve mice (24 h prior to TNFα). Right-hand panels show the same three time points after administration of liposomes 2 h after TNFα injection in the same mice. Liposome uptake increases after TNFα injection, but localization remains predominantly at the vessel margin, despite a massive influx of circulating leukocytes.

Fig. 4.

Flow cytometric analysis of cell types involved in immunoliposome uptake. Flow cytometry was performed on disaggregated brains following injection of anti–VCAM-1 or IgG control immunoliposomes (20 mg/kg). CD31 and CD45 staining were used to identify four distinct cell populations: endothelial (CD45⁻CD31⁺), microglia/macrophages (CD45^Mid), leukocytes (CD45^Hi), and double-negative (CD45⁻CD31⁻) cells. Representative two-dimensional plots from naïve (A) and TNFα-injured mice (B) show identification of each cell type (Left), and histograms of far red staining, representing anti-VCAM antibody or IgG control positivity (Middle) or anti-VCAM liposome or IgG-liposome positivity (Right) in the EC gate. A minimum of 2 × 10³ ECs was counted for all brains. (C) The percentage of liposome-positive ECs and leukocytes in control and TNFα-injured mice (Left) and the MFI of each cell type (Right) are summarized. While only a small percentage of ECs stain positive for anti-VCAM liposomes in control mice, more than half of recovered ECs are liposome-positive in TNFα-injured mice (*P < 0.001). A similar pattern is seen for the MFI, which is significantly higher in TNFα-injected vs. control mice (**P < 0.001). Each bar represents n = 3 mice with mean ± SD shown, and brains were collected 30 min after injection.

Fig. 5.

Targeted LNPs accumulate in the brain and induce TM expression via mRNA delivery. (A) Ipsilateral brain uptake at 30 min after injection of ¹²⁵I-labeled anti–VCAM-1- and control IgG-LNPs (8 µg of mRNA) in healthy and intrastriatal TNFα-injured mice. Tissue uptake is indicated as mean ± SEM (n = 3); tissue uptake of anti–VCAM-1-LNP was compared to IgG counterpart in both naïve and TNFα-treated mice (***P < 0.001, one-way ANOVA, Bonferroni post hoc). Anti–VCAM-1-LNP uptake was also compared in TNFα-treated mice vs. naïve mice (^###P < 0.001, one-way ANOVA, Bonferroni post hoc). Firefly luciferase expression (B) was measured in the ipsilateral hemisphere at 5 h after i.v. administration of anti–VCAM-1 and anti–ICAM-1-LNP-fLuc mRNAs in TNFα-injured mice (dashed line indicates firefly luciferase luminescence induced by IgG-LNP-fLuc mRNA). Luciferase transfection activity of anti–VCAM-1-LNP was compared to anti–ICAM-1-LNP (***P < 0.001, one-way ANOVA, Bonferroni post hoc). (C) Western blot for brain homogenates (10 μg total protein per lane) with staining for FLAG, TM, and α-actin. TNFα-injured mice (n = 4) were treated with anti–VCAM-1 (i.v. injected) and anti–ICAM-1 (administered via internal carotid artery) targeted LNPs bearing mRNA encoding for TM-FLAG (LNP-TM) 4.5 h after LNP treatment. (D) Normalized brain uptake (%ID/g in TNF-injured brain − %ID/g in naïve brain) of mAB against TM (5 µg of clone 411) 30 min after injection in TNF-injured animals (17 h after injury and 4.5 h after treatment with saline (Untreated) or VCAM/LNP-TM) (mean + SD; n = 4, t test: ***P < 0.001).

Fig. 6.

Brain edema: extravascular radiolabeled albumin accumulation in the brain. (A) Assessment of brain edema using albumin leakage assay. Radiolabeled albumin was injected 21 h after unilateral striatal injection of TNFα (0.5 μg) and allowed to circulate for 4 h. The ratio between extravasated and bloodstream radiolabeled albumin was determined as counts per minute per gram of brain:counts per minute per gram of blood. Treatment with TNFα (n = 14) significantly increased albumin leakage in the ipsilateral hemisphere (***P < 0.001 in sham ipsilateral vs. TNF ipsilateral and ^###P < 0.001 contralateral vs. ipsilateral in TNF injured and P < 0.001 TNF ipsilateral vs. naïve animals [n = 6], one-way ANOVA, Bonferroni post hoc). Data shown as mean ± SEM. (B) Sham normalized ablumin leakage, considering a 100% leakage as the value calculated for the ipsilateral hemisphere in PBS-treated animals and 0% leakage as the value calculated for the contralateral or ipsilateral hemisphere of the sham-operated animals. Treatment with anti–VCAM-1-targeted LNP-TM (n = 6) significantly reduced albumin leakage in ipsilateral hemisphere compared to nontargeted LNP-TM– (n = 5) and PBS vehicle- (n = 8) treated animals (***P < 0.001 vs. vehicle, one-way ANOVA, Bonferroni post hoc). Data shown as mean ± SEM.