Development of a Nanoparticle-Based Approach for the Blood-Brain Barrier Passage in a Murine Model of Amyotrophic Lateral Sclerosis

Abstract

The development of nanoparticles (NPs) to enable the passage of drugs across blood-brain barrier (BBB) represents one of the main challenges in neuropharmacology. In recent years, NPs that are able to transport drugs and interact with brain endothelial cells have been tested. Here, we investigated whether the functionalization of avidin-nucleic-acid-nanoassembly (ANANAS) with apolipoprotein E (ApoE) would allow BBB passage in the SOD1^G93A mouse model of amyotrophic lateral sclerosis. Our results demonstrated that ANANAS was able to transiently cross BBB to reach the central nervous system (CNS), and ApoE did not enhance this property. Next, we investigated if ANANAS could improve CNS drug delivery. To this aim, the steroid dexamethasone was covalently linked to ANANAS through an acid-reversible hydrazone bond. Our data showed that the steroid levels in CNS tissues of SOD1^G93A mice treated with nanoformulation were below the detection limit. This result demonstrates that the passage of BBB is not sufficient to guarantee the release of the cargo in CNS and that a different strategy for drug tethering should be devised. The present study furthermore highlights that NPs can be useful in improving the passage through biological barriers but may limit the interaction of the therapeutic compound with the specific target.

Keywords: Nanomedicine; amyotrophic lateral sclerosis; blood–brain barrier; pharmacology.

Figure 1

Schematic representation (not in scale of the formulations selected for the in vivo studies). Upper panel: components of the core ANANAS; lower panel: functional assemblies are generated by mixing core ANANAS with the different biotin components at predefined biotin:ANANAS BBS molar ratios.

Figure 2

Experimental plan for NP functionalization and delivery study (A) WT and SOD1^G93A mice (n = 12 mice per group) were intravenously (IV) treated with ANANAS, ANANAS-ApoE, or vehicle for ANANAS functionalization study. Liver (Li.), spinal cord (Sc.), and brain (Br.) were collected 30 min, 4 h and 24 h post treatment for biochemical and imaging analysis. (B) SOD1^G93A mice (n = 12 mice per group) were intravenously (IV) treated with ANANAS-Dex or Dex for ANANAS drug delivery study. Organs were collected for PK measurements 15-, 30-, and 60-min post treatment.

Figure 3

NP biodistribution in WT mice. (A) Avidin quantification from liver, brain, and spinal cord of WT mice intravenously treated with ANANAS and sacrificed 30 min after injection, with (pink bars) or (red bars) without intracardial perfusion. Data are mean ± SEM (n = three in each experimental group) and indicates the relative immunoreactivity (RI) normalized to total protein loading; * p < 0.05 versus the respective perfused tissue by Student’s t-test. (B–D) Dot blot analysis for avidin quantification in liver (B), brain (C), and spinal cord (D) of WT mice treated with vehicle (dotted line), ANANAS (red line), and ANANAS-ApoE (green line) and sacrificed with intracardial perfusion 30 min, 4 h, and 24 h after the injection. (B–D) Data are mean ± SD (n = three or four in each experimental group) and indicates the relative immunoreactivity (RI) normalized to total protein loading; * p < 0.05 versus vehicle; # p < 0.05 versus ANANAS-ApoE and § p < 0.05 versus ANANAS 30 min, by one-way ANOVA, Tukey’s post hoc test.

Figure 4

NP biodistribution in SOD1^G93A mice. (A–C) Dot blot analysis for avidin quantification in the liver (A), brain (B), and spinal cord (C) of SOD1^G93A mice treated with a vehicle (dotted line), ANANAS (red line), and ANANAS-ApoE (green line) and sacrificed with intracardial perfusion 30 min, 4 h, and 24 h after the injection. (A–C) Data are mean ± SD (n = three or four in each experimental group) and indicates the relative immunoreactivity (RI) normalized to total protein loading; * p < 0.05 versus vehicle; # p < 0.05 versus ANANAS-ApoE and § p < 0.05 versus 30 min, by one-way ANOVA, Tukey’s post hoc test.

Figure 5

Ex vivo biodistribution in SOD1^G93A mice. (A,B) Ex vivo optical analysis of brain and spinal cord from SOD1^G93A mice treated with saline (Vehicle) or with ANANAS and sacrificed with intracardial perfusion 30 min and 24 h after the treatment. (A) Representative images of brain and cervical (C.), lumbar (L.), spinal cord. (B) Quantification of ex vivo optical imaging signal in brain and spinal cord. Dotted lines indicate the mean of vehicle. Data (mean ± SD; n = three or four in each experimental group) indicates the relative radian efficiency and are expressed as percentages of the respective vehicle. * p < 0.05 versus vehicle, by one-way ANOVA, Tukey’s post hoc test.

Figure 6

Pharmacokinetics study of free Dex in SOD1^G93A mice. Levels of free Dex measured in plasma (A) and liver, brain, and spinal cord (B) from SOD1^G93A mice at different time points (15, 30, 60 min) after intravenous administration of ANANAS-Dex. Dex released from the NP was measured by HPLC MS/MS with limit of quantitation (LoQ) equal to 0.01 ng/g for the brain and spinal cord, and 0.1 ng/mL for plasma. Data are mean ± SEM (n = four in each experimental group); * p < 0.05 by one-way ANOVA, Tukey’s post hoc test. n.d.: not detected.