A Nanomule Peptide Carrier Delivers siRNA Across the Intact Blood-Brain Barrier to Attenuate Ischemic Stroke

Abstract

The blood-brain barrier (BBB) hinders the distribution of therapeutics intended for treatment of neuroinflammation (NI) of the central nervous system. A twelve-amino acid peptide that transcytoses the BBB, termed MTfp, was chemically conjugated to siRNA to create a novel peptide-oligonucleotide conjugate (POC), directed to downregulate NOX4, a gene thought responsible for oxidative stress in ischemic stroke. The MTfp-NOX4 POC has the ability to cross the intact BBB and knockdown NOX4 expression in the brain. Following induction of ischemic stroke, animals pretreated with the POC exhibited significantly smaller infarcts; accompanied by increased protection against neurological deterioration and improved recovery. The data demonstrates that the MTfp can act as a nanomule to facilitate BBB transcytosis of siRNAs; where the NOX-4 specific siRNA moiety can elicit effective therapeutic knockdown of a gene responsible for oxidative stress in the central nervous system. This study is the first to conclusively demonstrate both siRNA-carrier delivery and therapeutic efficacy in any CNS disease model where the BBB remains intact and thus offers new avenues for potential treatments of oxidative stress underlying neuroinflammation in a variety of neuropathologies that are currently refractory to existing therapies.

Keywords: MTfp; NOX4; blood-brain barrier; peptide-oligonucleotide conjugate; siRNA; stroke.

FIGURE 1

Design of the MTfp-RNA conjugates. SMCC, succinimidyl 4-(N- maleimidomethyl) cyclohexane-1-carboxylate.

FIGURE 2

Conjugation to MTfp enables siRNA to cross an intact BBB and into the brain parenchyma. Representative 3D confocal images showing localization of MTfp-siRNA in the brain of mice with intact BBB. Cell nuclei are blue (DAPI) and capillaries are green (Tomato lectin-FITC). (A) AF680 fluorescence (red) in the cerebral cortex sections from a mouse treated with MTfp-siRNA_AF680; (B) Shows the enlarged area that has been surface-rendered to indicate the surface of the capillaries where FITC labelled capillaries (green) and MTfp-siRNA_AF680 (red); (C) Shows the enlarged area where MTfp-siRNA_AF680 is localized within the blood capillaries (yellow dots, see arrow); (D) AF680 fluorescence (red) in the mouse cerebral cortex treated with PBS (i.e., background fluorescence); (E) Control AF680 fluorescence (red) in the mouse cortex treated with siRNA_AF680. (F) Distribution of MTfp-siRNA in the cortex of wild type mice with intact BBB. Values indicate total AF680 fluorescence normalized to total tissue volume (VTA in Supplementary Table S1) and then normalized to the total AF680 fluorescence seen in PBS (background). Data are represented as means ± SD (n = 3, eight fields of view per animal). *p-value < 0.05.

FIGURE 3

NOX4 mRNA expression in the brain after siRNA treatment. The graphs show the fold change of Nox4 gene expression in the mouse brain hemispheres measured by qPCR following treatment and 24 h post-stroke, showing both the expression in left ischemic side and the right contralateral side. (A) Values are normalized to β-actin gene expression and individual samples are compared to the corresponding average PBS-sham condition (ischemic or contralateral). Data are shown as mean ± SEM. (n = 2–4 mice per group). (B) Ratio of average fold change of each group compared to PBS-sham. (C) Ratio of average fold change of each group compared to PBS-stroke. “Left ischemic” in blue, refers to the measurments in the left stroke-induced hemisphere and “Right contralateral” in orange, refers to the measurement in the right non-stroke hemisphere. Raw data and calculations are shown in Supplementary Table S3.

FIGURE 4

Top panel. (A – C): Treatment with MTfp-siRNA confers neuroprotection and reduces damage after ischemic stroke. (A) TTC-stained brain sections of mice receiving various IV injections, followed by ischemic stroke. Tissue was collected 24 h after surgery (B) Infarct volume was quantitated by measuring absorbance of solvent extracted dye (* p < 0.05, one-way ANOVA). (C) Neuroscore at 0.5 and 24 h after stroke induction in mice pretreated with siRNA, MTfp-siRNA or PBS control. *p < 0.05, **p < 0.001. Bottom Panel (D–F): Treatment with MTfp-scrambled RNA does not offer neuroprotection after ischemic stroke. (D) TTC-stained brain sections of mice receiving various IV injections, followed by ischemic stroke. (E) Infarct volume was quantitated by measuring absorbance (485 nm) of solvent extracted dye. Data are shown as mean ± SEM (no significant difference). (F) Neuroscore at 0.5 and 24 h after stroke induction. Data are shown as mean ± SEM (There is no significant difference between the treatments at either time point) (n = 3 mice per group).