Alanine and glutathione targeting of dopamine- or ibuprofen-coupled polypeptide nanocarriers increases both crossing and protective effects on a blood-brain barrier model

Abstract

Background: Targeting the blood-brain barrier (BBB) is a key step for effective brain delivery of nanocarriers. We have previously discovered that combinations of BBB nutrient transporter ligands alanine and glutathione (A-GSH), increase the permeability of vesicular and polypeptide nanocarriers containing model cargo across the BBB. Our aim was to investigate dopamine- and ibuprofen-coupled 3-armed poly(L-glutamic acid) nanocarriers targeted by A-GSH for transfer across a novel human co-culture model with induced BBB properties. In addition, the protective effect of ibuprofen containing nanoparticles on cytokine-induced barrier damage was also measured.

Method: Drug-coupled nanocarriers were synthetized and characterized by dynamic light scattering and transmission electron microscopy. Cellular effects, uptake, and permeability of the nanoparticles were investigated on a human stem cell-based co-culture BBB model with improved barrier properties induced by a small molecular cocktail. The model was characterized by immunocytochemistry and permeability for marker molecules. Nanocarrier uptake in human brain endothelial cells and midbrain organoids was quantified by spectrofluorometry and visualized by confocal microscopy. The mechanisms of cellular uptake were explored by addition of free targeting ligands, endocytic and metabolic inhibitors, co-localization of nanocarriers with intracellular organs, and surface charge modification of cells. The protective effect of ibuprofen-coupled nanocarriers was investigated against cytokine-induced barrier damage by impedance and permeability measurements.

Results: Targeted nanoformulations of both drugs showed elevated cellular uptake in a time-dependent, active manner via endocytic mechanisms. Addition of free ligands inhibited the cellular internalization of targeted nanocarriers suggesting the crucial role of ligands in the uptake process. A higher permeability across the BBB model was measured for targeted nanocarriers. After crossing the BBB, targeted dopamine nanocarriers subsequently entered midbrain-like organoids derived from healthy and Parkinson's disease patient-specific stem cells. The ibuprofen-coupled targeted nanocarriers showed protective effects against cytokine-induced barrier damage.

Conclusion: BBB-targeted polypeptide nanoparticles coupled to therapeutic molecules were effectively taken up by brain organoids or showing a BBB protective effect indicating potential applications in nervous system pathologies.

Keywords: Alanine; Blood–brain barrier; Dopamine; Dual-targeted nanocarriers; Glutathione; Human stem cell derived endothelial cell; Ibuprofen; In vitro model; Poly(l-glutamic acid).

Fig. 1

Synthesis of nanocarriers. a Synthesis and debenzylation of poly(γ-benzyl-l-glutamic acid) 3-PBLG. b Synthesis of copolypeptides using EDC/NHS coupling, and c modification of ibuprofen (ibu)

Fig. 2

The human BBB co-culture model. a Signaling pathways related to BBB maturation induced by cARLA. b Claudin-5 immunostaining of human stem cell-derived brain endothelial cells. Blue: nuclei; red: claudin-5, scale bar: 20 µm. c Expression levels of genes encoding alanine transporters in the cARLA treated human BBB model. Values presented are means ± SD

Fig. 3

Characterization of nanocarriers. a Schematic drawing of non-targeted, 3-armed poly(L-glutamic acid) nanocarriers grafted with dopamine (3-PLG-dopa) or ibuprofen (3-PLG-ibu) and their L-alanine (A) and glutathione (GSH) dual-targeted (3-PLG-dopa-A-GSH; 3-PLG-ibu-A-GSH) formulations. The copolypeptides were labeled by rhodamine 6G (R6G). b The main physico-chemical properties of nanocarriers. Values presented are means ± SD. c Transmission electron microscopy images of nanoformulations. Scale bar: 100 nm

Fig. 4

Cellular uptake of a dopamine- (3-PLG-dopa; 3-PLG-dopa-A-GSH) or b ibuprofen-coupled (3-PLG-ibu; 3-PLG-ibu-A-GSH) nanocarriers in brain endothelial cells after 1, 4 and 24 h of incubation (100 µg/ml; 37 °C) and the effect of free l-alanine and glutathione (GSH) ligands (5 mM each in co-treatment with nanocarriers) on the cellular internalization of dual-targeted nanocarriers. Values presented are means ± SD and given as a percentage of the 3-PLG-dopa or 3-PLG-ibu groups at 1 h-time point. Statistical analysis: two-way ANOVA, Tukey’s post-test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared to the 3-PLG-dopa-A-GSH or 3-PLG-ibu-A-GSH groups at each time-points; ^#p < 0.05; ^##p < 0.01; ^###p < 0.001 between the 3-PLG-dopa-A-GSH or 3-PLG-ibu-A-GSH groups at each time point; n = 6

Fig. 5

Live cell visualization of cellular uptake. Confocal microscopy images of living brain endothelial cells after 24-h incubation with a dopamine- (3-PLG-dopa; 3-PLG-dopa-A-GSH) or b ibuprofen-coupled (3-PLG-ibu; 3-PLG-ibu-A-GSH) nanoparticles. Nanocarriers: yellow; cell nuclei: cyan; scale bar: 20 μm. Image analysis of cellular entry of c dopamine- or d ibuprofen-coupled nanocarriers. Values are presented as means ± SD, and shown as percentage of the untreated background fluorescent intensity given as arbitrary units (a.u.). Statistical analysis: one-way ANOVA, Tukey’s post-test; ****p < 0.0001 compared to the 3-PLG-dopa or 3-PLG-ibu groups; ^####p < 0.0001 compared to the background; n = 4–10

Fig. 6

Mechanisms of nanocarrier cell entry. The effects of endocytosis inhibitors randomly methylated β-cyclodextrin (CD) or cytochalasin D (CytoD) and metabolic inhibitor sodium azide on the uptake of a 3-PLG-dopa and 3-PLG-dopa-A-GSH, and b 3-PLG-ibu and 3-PLG-ibu-A-GSH nanocarriers. c Schematic drawing of the modification of brain endothelial surface charge by neuraminidase (NA) enzyme or cationic lipid TMA-DPH. The effect of NA and TMA-DPH on the cellular uptake of d 3-PLG-dopa and 3-PLG-dopa-A-GSH and e 3-PLG-ibu and 3-PLG-ibu-A-GSH nanocarriers. Values presented are means ± SD and are given as a percentage of the non-targeted nanoparticle groups. Statistical analysis: two-way ANOVA, Dunnett post-test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared to the non-treated control in each groups; ^####p < 0.0001 compared to non-targeted groups. n = 4–6

Fig. 7

Permeability of nanocarriers across the human co-culture model of the BBB. a Schematic drawing of the experimental set-up. Permeability of Evans blue-albumin (EBA), sodium fluorescein (SF) reference marker molecules and b 3-PLG-dopa and 3-PLG-dopa-A-GSH and c 3-PLG-ibu and 3-PLG-ibu-A-GSH nanocarriers across the human BBB co-culture model. Values are means ± SD. Statistical analysis: one-way ANOVA followed by Dunnett test. **p < 0.01; ****p < 0.0001 compared to the 3-PLG-dopa or 3-PLG-ibu groups. n = 4–6. P_app: apparent permeability coefficient

Fig. 8

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

Fig. 9

Representative confocal fluorescent microscopy images showing the uptake of non-targeted (3-PLG-dopa) and alanine-glutathione-targeted (3-PLG-dopa-A-GSH) nanocarriers (yellow) by one control and one PD midbrain-specific organoids after crossing the BBB model. Cell nuclei are stained by Hoechst 33342 (cyan). Scale bar: 200 μm

Fig. 10

Protective effects of ibuprofen-coupled nanocarriers (3-PLG-ibu and 3-PLG-ibu-A-GSH) or ibuprofen against cytokine-induced (CK: TNF-α + IL1-β) barrier dysfunction on human brain endothelial cells. a Cell response kinetics monitored by real-time impedance measurements for 24 h. b Impedance of human brain endothelial cells at the 24-h time point. Values presented are means ± SD and are given as normalized impedance. Statistical analysis: one-way ANOVA followed by Tukey’s post-test; ^aaaa p < 0.0001 compared to the control group; **p < 0.01, ****p < 0.0001, compared to the CK group; ^####p < 0.0001 between the nanocarrier and ibuprofen groups; n = 6–8

Fig. 11

Protective effects of ibuprofen-coupled nanocarriers (3-PLG-ibu and 3-PLG-ibu-A-GSH) and ibuprofen against cytokine-induced (CK) barrier dysfunction in a human BBB co-culture model. a schematic drawing of the set-up. b Effect of CK on the permeability of 3-PLG-ibu and 3-PLG-ibu-A-GSH nanocarriers across the BBB model. Values presented are means ± SD. Statistical analysis: two-way ANOVA followed by Tukey’s post-test; comparisons within the treatment groups ****p < 0.0001; comparisons between the control and the CK groups; ^#p < 0.05; ^####p < 0.0001; n = 4. P_app: apparent permeability coefficient. Penetration of c sodium fluorescein (SF) and d Evans blue-albumin (EBA) reference marker molecules after a 24-h permeability assay with nanocarriers or free ibuprofen (1.5 µM) with or without CK-treatment. Values presented are means ± SD. Statistical analysis: two-way ANOVA followed by Tukey’s post-test; comparisons within the treatment groups *p < 0.05; ****p < 0.0001; comparisons between the control and the CK groups; ^####p < 0.0001 n = 4. P_app: apparent permeability coefficient