Targeting Human Endothelial Cells with Glutathione and Alanine Increases the Crossing of a Polypeptide Nanocarrier through a Blood-Brain Barrier Model and Entry to Human Brain Organoids

Abstract

Nanoparticles (NPs) are the focus of research efforts that aim to develop successful drug delivery systems for the brain. Polypeptide nanocarriers are versatile platforms and combine high functionality with good biocompatibility and biodegradability. The key to the efficient brain delivery of NPs is the specific targeting of cerebral endothelial cells that form the blood-brain barrier (BBB). We have previously discovered that the combination of two different ligands of BBB nutrient transporters, alanine and glutathione, increases the permeability of vesicular NPs across the BBB. Our aim here was to investigate whether the combination of these molecules can also promote the efficient transfer of 3-armed poly(l-glutamic acid) NPs across a human endothelial cell and brain pericyte BBB co-culture model. Alanine and glutathione dual-targeted polypeptide NPs showed good cytocompatibility and elevated cellular uptake in a time-dependent and active manner. Targeted NPs had a higher permeability across the BBB model and could subsequently enter midbrain-like organoids derived from healthy and Parkinson's disease patient-specific stem cells. These results indicate that poly(l-glutamic acid) NPs can be used as nanocarriers for nervous system application and that the right combination of molecules that target cerebral endothelial cells, in this case alanine and glutathione, can facilitate drug delivery to the brain.

Keywords: 3-armed polypeptides; alanine; blood–brain barrier; brain endothelial cells; brain organoid; dual-targeting; glutathione; peptide nanocarriers.

Figure 1

(a) Synthesis of 3-armed poly(l-glutamic acid γ-benzyl ester) (3-PBLG) and deprotection of benzyl groups to prepare 3-armed poly(l-glutamic acid) (3-PLG), (b) Synthesis of l-alanine (A) and glutathione (GSH)-targeted, 3-armed poly(l-glutamic acid) (3-PLG-A–GSH), (c) Synthesis of rhodamine 6G (R6G), labeled 3-PLG-R6G or 3-PLG-A–GSH-R6G.

Figure 2

¹H NMR spectra and structure of (a) 3-armed poly(γ-benzyl-l-glutamic acid (3-PBLG) in trifluoroacetic acid (TFA-d₁), (b) 3-armed poly(l-glutamic acid) (3-PLG) in deuterium oxide (D₂O), and (c) l-alanine and glutathione-targeted 3-armed poly(l-glutamic acid) (3-PLG-A-GSH) in D₂O.

Figure 3

Transmission electron microscopy images of (a) non-targeted (3-PLG) and (b) alanine–glutathione-targeted (3-PLG-A-GSH) nanocarriers. Scale bar: 100 nm.

Figure 4

Effect of non-targeted (3-PLG) and alanine–glutathione-targeted (3-PLG-A-GSH) nanocarriers on the viability of human brain endothelial cells. Impedance kinetics of cell responses to (a) 3-PLG and (b) 3-PLG-A-GSH monitored for 24 h by real-time measurements. Impedance of human brain endothelial cells incubated with (c) 3-PLG and (d) 3-PLG-A-GSH at the 24-h time point. (a–d) Values presented are means ± SD and are given as cell index. Effect of (e) 3-PLG and (f) 3-PLG-A-GSH on the cell viability at 24 h measured by MTT test. Values presented are means ± SD and are given as a percentage of the control group. Statistical analysis: one-way ANOVA followed by Dunnett’s post-test; * p < 0.05, **** p < 0.0001, compared to the control group; n = 6–8.

Figure 5

Cellular uptake of non-targeted (3-PLG) and alanine–glutathione-targeted (3-PLG-A-GSH) polypeptide nanocarriers in cultured human brain endothelial cells after 1, 4 and 24 h of incubation (100 µg/mL; 37 °C). Values presented are means ± SD and are given as a percentage of the 3-PLG group’s 1 h time point. Statistical analysis: two-way ANOVA; **** p < 0.0001 compared to the 3-PLG group at each time point; #### p < 0.0001 compared to the 3-PLG-A-GSH group at the 1 h time point; n = 6.

Figure 6

Confocal microscopy images showing the entry of (a) non-targeted (3-PLG) and (b) alanine–glutathione-targeted (3-PLG-A-GSH) nanocarriers labeled with R6G (red) into living human brain endothelial cells (100 µg/mL; 24 h incubation). Cell nuclei are stained with Hoechst 33,342 (blue). Scale bar: 20 μm. (c) Evaluation of the R6G fluorescence intensity of cells incubated with 3-PLG or 3-PLG-A-GSH (24 h). Values are means ± SD and given as arbitrary units (a.u.), shown as percentage of the 3-PLG group. Statistical analysis: unpaired t-test, **** p < 0.0001, compared to the 3 PLG group; n = 4.

Figure 7

Cellular uptake and mechanism of polypeptide nanocarrier cell entry. (a) Uptake of 3-PLG and 3-PLG-A-GSH nanocarriers in human brain endothelial cells (4 h; 50 µg/mL). Values are means ± SD and given as a percentage of the non-targeted 3-PLG data. Statistical analysis: unpaired t-test, **** p < 0.0001, compared to the 3 PLG group. The effect of low temperature (4 °C) and endocytosis inhibitor randomly methylated β-cyclodextrin (5 mM) on the uptake of (b) 3-PLG and (c) 3-PLG-A-GSH. Values are means ± SD and given as a percentage of the control group (37 °C data, no inhibition). Statistical analysis: one-way ANOVA, Dunnett test, * p < 0.05, ** p < 0.01 compared to the respective control groups of 3-PLG and 3-PLG-A-GSH treatments; n = 6.

Figure 8

Penetration of non-targeted (3-PLG) and alanine–glutathione-targeted (3-PLG-A-GSH) nanocarriers across the human co-culture model of the blood–brain barrier (24 h incubation, 50 µg/mL, 37 °C). (a) Schematic drawing of the experimental set-up. (b) Penetration of 3-PLG and 3-PLG-A-GSH across the blood–brain barrier model. Values are means ± SD and given as a percentage of the total nanocarrier amount in the upper, donor compartment at t = 0. Statistical analysis: unpaired t-test; **** p < 0.0001; n = 6.

Figure 9

Permeability of non-targeted (3-PLG) and alanine-glutathione-targeted (3-PLG-A-GSH) nanocarriers across the human co-culture model of the blood–brain barrier and entry into midbrain-specific organoids (24 h, 100 µg/mL, 37 °C). (a) Schematic drawing of the experimental set-up. (b) Permeability of Evans blue-albumin (EBA) and sodium fluorescein (SF) reference marker molecules across the BBB model. Permeability of 3-PLG and 3-PLG-A-GSH across the co-culture model in the presence of midbrain-specific organoids derived from healthy control and from Parkinson’s disease (PD) patients’ cells. (c) Cellular uptake of 3-PLG and 3-PLG-A-GSH by organoids after crossing the blood–brain barrier. Values are means ± SD, n = 6. Statistical analysis: two-way ANOVA, Dunnett test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to the 3-PLG data in both organoid groups; # p < 0.05 compared between organoid groups. Permeability values of EBA and SF were compared to the 3-PLG group with control organoids (*** p < 0.001, **** p < 0.0001; one-way ANOVA, Dunnett test). P_app: apparent permeability coefficient.

Figure 10

Representative confocal fluorescent microscopy images showing the uptake of non-targeted (3-PLG) and alanine–glutathione-targeted (3-PLG-A-GSH) nanocarriers (red) by midbrain-specific organoids derived from healthy (control) patients and from Parkinson’s disease (PD) patients’ cells after crossing the blood–brain barrier model (24 h, 37 °C). Cell nuclei are stained by Hoechst 33342 (blue). Scale bar: 200 μm.