A Triple Combination of Targeting Ligands Increases the Penetration of Nanoparticles across a Blood-Brain Barrier Culture Model

Abstract

Nanosized drug delivery systems targeting transporters of the blood-brain barrier (BBB) are promising carriers to enhance the penetration of therapeutics into the brain. The expression of solute carriers (SLC) is high and shows a specific pattern at the BBB. Here we show that targeting ligands ascorbic acid, leucine and glutathione on nanoparticles elevated the uptake of albumin cargo in cultured primary rat brain endothelial cells. Moreover, we demonstrated the ability of the triple-targeted nanovesicles to deliver their cargo into midbrain organoids after crossing the BBB model. The cellular uptake was temperature- and energy-dependent based on metabolic inhibition. The process was decreased by filipin and cytochalasin D, indicating that the cellular uptake of nanoparticles was partially mediated by endocytosis. The uptake of the cargo encapsulated in triple-targeted nanoparticles increased after modification of the negative zeta potential of endothelial cells by treatment with a cationic lipid or after cleaving the glycocalyx with an enzyme. We revealed that targeted nanoparticles elevated plasma membrane fluidity, indicating the fusion of nanovesicles with endothelial cell membranes. Our data indicate that labeling nanoparticles with three different ligands of multiple transporters of brain endothelial cells can promote the transfer and delivery of molecules across the BBB.

Keywords: ascorbic acid; blood-brain barrier; brain endothelial cell; brain organoid; drug targeting; glutathione; leucine; nanoparticle; solute carrier; triple targeting ligand.

Figure 1

(a) Schematic drawing of non-targeted (N) and ascorbic acid-glutathione-leucine targeted (N-AGL) nanoparticles. TR-BSA: Texas red-labeled bovine serum albumin. (b) TEM images of nanoparticles. Bar: 100 nm. (c) Main physico-chemical properties of the nanoparticles. Values presented are means ± SD.

Figure 2

Effect of non-targeted (N) and ascorbic acid-glutathione-leucine-targeted (N-AGL) nanoparticles on the impedance of primary rat brain endothelial cells monitored by real-time measurements. Kinetics of brain endothelial cell responses to (a) N and (b) N-AGL nanoparticle treatments for 24 h. Values presented are means ± SD and are given as cell index. Impedance of brain endothelial cells treated with N (c) or N-AGL (d) after 4 or 8 h. Values presented are means ± SD and are given as a percentage of culture medium-treated control. Statistical analysis: one-way ANOVA followed by Dunnett’s post-test; * p < 0.05, ** p < 0.01, compared to the control group; n = 6–8.

Figure 3

Cellular uptake of Texas red labeled albumin (TR-BSA), and TR-BSA loaded into non-targeted (N) and ascorbic acid-glutathione-leucine-targeted (N-AGL) nanoparticles in cultured primary rat brain endothelial cells after (a) 4 h and (b) 8 h of incubation. Values presented are means ± SD and are given as a percentage of the TR-BSA group at both time points. Statistical analysis: unpaired t-test; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the TR-BSA groups; n = 6. (c) Effect of temperature, endocytic inhibitors filipin (5 µg/mL) and cytochalasin D (CD; 0.125 µg/mL) or metabolic inhibitor sodium azide (azide; 1 mg/mL) on the cellular uptake of TR-BSA loaded N-AGL (4 h, 37 °C). Values presented are means ± SD and are given as a percentage of the control group (C; at 37 °C without treatment). Statistical analysis: one-way ANOVA followed by Dunnett’s posttest; ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to the control group; n = 6. (d) The effect of neuraminidase (Neu, 1 U/mL) and TMA-DPH (30 mM) on the uptake of TR-BSA loaded N-AGL in rat brain endothelial cells. Values presented are means ± SD and are given as a percentage of the control group. Statistical analysis: one-way ANOVA followed by Dunnett’s posttest; * p < 0.05 compared to the control group; n = 6.

Figure 4

(a) Confocal microscopy images of cultured RBECs incubated with Texas red-labeled albumin (TR-BSA; red fluorescence), non-targeted (N) or ascorbic acid-glutathione-leucine-targeted (N-AGL) nanoparticles. Cell nuclei are shown in blue. Scale bar: 50 μm. (b) Fluorescence intensity evaluation of TR-BSA, N or N-AGL treated cells. (24 h, 37 °C). Values presented are means ± SD and are given as arbitrary units (a.u.), shown as percentage of the TR-BSA group. Statistical analysis: one-way ANOVA followed by Dunnett’s posttest; * p < 0.05, ** p < 0.01, compared to the TR-BSA group; n = 6.

Figure 5

Plasma membrane fluidity measured by fluorescence anisotropy in living brain endothelial cells treated with non-targeted (N) or ascorbic acid-glutathione-leucine-targeted (N-AGL) nanoparticles and membrane fluidizer benzyl alcohol (30 mM). Values presented are means ± SD. Statistical analysis: two-way ANOVA, Bonferroni posttest. ^# p < 0.05, all groups were compared to medium treated control (C); **** p < 0.0001, compared to first column of each group, n = 3.

Figure 6

Permeability of the cargo Texas red-BSA (TR-BSA) alone and encapsulated into non-targeted (N) and ascorbic acid-glutathione-leucine-targeted (N-AGL) nanoparticles across the BBB co-culture model. Papp: apparent permeability coefficient. Values presented are means ± SD. Statistical analysis: one-way ANOVA followed by Dunnett’s posttest; * p < 0.05 compared to the TR-BSA group; n = 4.

Figure 7

Entry of ascorbic acid-glutathione-leucine-targeted, Texas Red-BSA (TR-BSA; red) loaded nanoparticles (N-AGL) into midbrain organoids after passage across the BBB model. (a) Schematic drawing of the experiment. (b) Representative confocal microscopy images of midbrain-specific organoids derived from Parkinson’s disease patients (PD) and healthy control (Control). Neurons were immunostained with βIII-tubulin (green), and cell nuclei were stained with bis-benzimide (blue). Bar: 100 μm. (c) Fluorescence intensity evaluation of the red channel of the images of control and PD midbrain-like organoids indicating TR-BSA entry. Values presented are means ± SD and are given as arbitrary units (a.u.). Statistical analysis: Student’s t-test; ** p < 0.01, n = 8–10.