Targeting lipid nanoparticles to the blood-brain barrier to ameliorate acute ischemic stroke

Abstract

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.

Keywords: drug delivery; drug targeting; ischemic stroke; lipid nanoparticles; neurovascular inflammation.

Graphical abstract

Figure 1

In the tMCAO model of AIS, CAM-targeted antibodies and NCs accumulate in the brain at vastly higher levels (A) Timeline for all biodistribution experiments. Mice were challenged with ischemia (45 min) and reperfusion, followed by IV injection of ¹²⁵I-radiolabeled mAbs/liposomes/LNPs or untargeted IgG controls 24 h after injury, and tissue biodistribution was determined 30 min after injection. (B, F, and J) Schematic representation of radiolabeled mAbs, mAb-conjugated liposomes, mAb-conjugated LNPs, respectively. (C, G, and K) All anti-CAM mAbs/liposomes/LNPs had markedly higher brain uptake than control IgG counterpart, with the highest uptake in the ipsilateral (injured) hemisphere being anti-VCAM. Anti-ICAM mAb was the only mAb that showed a statistically significant preference for accumulation in the ipsilateral vs. contralateral hemisphere. ∗p < 0.05, ∗∗p < 0.01, ∗p < 0.001, ∗∗∗∗p < 0.0001 using two-way ANOVA followed by Dunnett’s post hoc against IgG, #p < 0.05, ##p < 0.01 using paired t-test comparing contralateral vs. ipsilateral. See also Tables S2–S4. (D, H, and L) The brain-to-blood distribution ratio in ipsilateral tMCAO brain further illustrates the superiority of VCAM-targeted mAbs/liposomes/LNPs. The values observed in naive mice are shown by dashed lines. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 using one-way ANOVA followed by Tukey’s post-hoc against IgG control. (E) ISI (localization ratio targeted mAb/localization ratio untargeted IgG) of mAbs/liposomes/LNPs in tMCAO mice showed significant lung targeting of ICAM over other organs, non-significant lung targeting of PECAM over other organs, and non-significant higher targeting of VCAM when compare spleen vs. brain. See also Figure S3. n = 3. ∗p < 0.05, ∗∗p < 0.01, ∗p < 0.001, ∗∗∗∗p < 0.0001 using two-way ANOVA followed by Tukey’s post-hoc. %p < 0.05, +p < 0.05 using one-way ANOVA followed by Dunnett’s post hoc against lungs, and #p < 0.5 against spleen. Data are presented as mean ± SEM.

Figure 2

VCAM-targeted NCs localize to endothelial cells and leukocytes in tMCAO brains and concentrate cargos in ipsilateral hemisphere (A–H) tMCAO mice were IV-injected with fluorophore-labeled VCAM-targeted liposomes, and single-cell suspensions were prepared from the brains 30 min later. Flow cytometry of the ipsilateral hemisphere shows higher VCAM-targeted liposome uptake in endothelial cells (CD31⁺/CD45^–; [A] ipsilateral, [B] contralateral) and leukocytes (CD45⁺; [E] ipsilateral, [F] contralateral), compared with uptake in the contralateral hemisphere. The contralateral hemisphere had fewer total leukocytes infiltrate into the brain, compared with the ipsilateral hemisphere (E–F and H). Comparison of the contralateral (black) and ipsilateral (red) hemispheres in terms of the number of recovered endothelial cells (C) and leukocytes (G) that were liposome positive showed a trend toward more liposome-positive cells in the ipsilateral hemisphere, for both cell types, with ∗p < 0.05 using Student’s t-test. Comparison of liposome fluorescence intensity in endothelial cells (D) and leukocytes (H) of the ipsilateral (red) vs. contralateral (black) brain showed higher liposome uptake in both cell types in the ipsilateral brain. ∗∗∗∗p < 0.0001 using Kolmogorov-Smirnov test. n = 4. (I) Cresyl violet staining of brain slices from tMCAO mice treated with or without VCAM-targeted liposomes loaded with dexamethasone (dex), from untreated tMCAO mice, and from naive mice. MSI of dex in the consecutive slices. (J) Whole-brain CLARITY images in Ai9 mice show leukocytes (CD45, blue), cells transfected by VCAM-targeted LNP/Cre-recombinase mRNA treatment (tdTomato, red), and endothelial cells (CD31, green). Scale bar, 3,000 μm. (K) Confocal image of brain from tMCAO mTmG mice injected with VCAM-targeted LNP-Cre-recombinase mRNA. Transfected cells switch from tdTomato to GFP (Figure S6A). Dashed line split the core/penumbra area and the arrow indicates the core of injury. Endothelial cells (CD31⁺) were transfected in both infarct core and penumbra (top), and infiltrated leukocytes (CD45⁺) in the infarct core were transfected (bottom). Scale bar, 100 μm.

Figure 3

VCAM-targeted NCs improved stroke outcomes (A) Mice were treated with VCAM-targeted NCs loaded with the anti-inflammatory drug dexamethasone (Dex) or IL-10 mRNA, or various controls. Each animal received three IV injections: one immediately after tMCAO reperfusion, then two doses every 24 h thereafter. Mice were euthanized at 72 h and the infarct volume was measured postmortem by staining for dead brain tissue. (B) Among the survived animals, VCAM-targeted liposomes loaded with Dex reduced stroke volume at 72 h, relative to all controls. Neither free Dex (no NC) nor empty VCAM-targeted liposomes decreased stroke volume. Dashed line: infarct volume at 24 h after tMCAO. (C) Dex-loaded VCAM targeted liposomes showed a higher but non-significant survival rate. For (B) and (C), n = 10 for control and n = 6 for the treatment groups, due to additional control in each cohort. (D) Luciferase expression in the naive or ipsilateral tMCAO brain showed elevated luciferase level after IV injection of VCAM-targeted LNP, compared with untargeted IgG control, and was improved especially in tMCAO brains. ∗∗p < 0.01, ∗∗∗∗p < 0.0001 using one-way ANOVA followed by Tukey’s post hoc. n = 4. (E) IL-10 mRNA-loaded VCAM targeted LNP reduced stroke volume by 62.1%, while untargeted control (bare) LNP had no significant treatment effect. n = 10 for untreated control, n = 8 for control LNP/IL-10 and VCAM-targeted LNP/IL-10. ∗p < 0.05 using one-way ANOVA followed by Dunnett’s post hoc. (F) IL-10 expression was detected in endothelial cells in tMCAO brain ipsilateral hemisphere with VCAM LNP/IL-10 mRNA treatment, but not with control LNP/IL-10 mRNA. Full panel shown in Figure S14. Scale bar, 100 μm. (G) IL-10 mRNA-loaded VCAM targeted LNP significantly improved the survival rate. ∗p < 0.05 using a log rank test. (H) Elevation of plasma IL-10 concentration following treatment LNP loaded with IL-10 mRNA. n = 4. ∗∗∗∗p < 0.0001 using Student’s t-test. (I) VCAM LNP/IL-10 treatment leads to significant reduction of CD45⁺ cells and macrophage/microglia. n = 3. ∗∗∗∗p < 0.0001 using two-way ANOVA followed by Sidak’s test. Data are presented as mean ± SEM.