The effect of drug loading and multiple administration on the protein corona formation and brain delivery property of PEG-PLA nanoparticles

Abstract

The presence of protein corona on the surface of nanoparticles modulates their physiological interactions such as cellular association and targeting property. It has been shown that α-mangostin (αM)-loaded poly(ethylene glycol)-poly(l-lactide) (PEG-PLA) nanoparticles (NP-αM) specifically increased low density lipoprotein receptor (LDLR) expression in microglia and improved clearance of amyloid beta (Aβ) after multiple administration. However, how do the nanoparticles cross the blood‒brain barrier and access microglia remain unknown. Here, we studied the brain delivery property of PEG-PLA nanoparticles under different conditions, finding that the nanoparticles exhibited higher brain transport efficiency and microglia uptake efficiency after αM loading and multiple administration. To reveal the mechanism, we performed proteomic analysis to characterize the composition of protein corona formed under various conditions, finding that both drug loading and multiple dosing affect the composition of protein corona and subsequently influence the cellular uptake of nanoparticles in b.End3 and BV-2 cells. Complement proteins, immunoglobulins, RAB5A and CD36 were found to be enriched in the corona and associated with the process of nanoparticles uptake. Collectively, we bring a mechanistic understanding about the modulator role of protein corona on targeted drug delivery, and provide theoretical basis for engineering brain or microglia-specific targeted delivery system.

Keywords: Aβ, amyloid beta; BBB, blood‒brain barrier; Brain delivery; CNS, central nervous system; DLS, dynamic light scattering; HCS, high content screening; LDLR, low density lipoprotein receptor; MCI, mild cognitive impairment; Microglia; NP, blank PEG-PLA nanoparticles; NP-corona complexes, nanoparticle-corona complexes; NP-αM, αM-loaded PEG-PLA nanoparticles; Nanoparticles; PEG-PLA; PEG-PLA, poly(ethylene glycol)–poly(l-lactide); Protein corona; TEER, trans-epithelial electrical resistance; TEM, transmission electronic microscope; cou6, coumarin 6; cou6-NPs, cou6-labeled nanoparticles; cou7, coumarin 7; αM, α-mangostin.

Graphical abstract

Figure 1

Brain distribution of PEG-PLA nanoparticles under different loading condition and dosing frequency. (A) Schematic of the nanoparticles administered into C57/BL6 mice (70 mg/kg, i.v.). Microglial cells were isolated by using magnetic beads. The concentration of cou6-NPs in blood, brain and microglial cells was determined by HPLC. The protein content of brain and microglial cells was determined by BCA Protein Assay. (B and C) The concentrations of cou6-NPs in blood (B) and brain (C) at 20, 60 and 100 min after administration. (D) Brain/blood ratio of cou6-NPs at 60 min. (E) The ratio of cou6-NPs in microglial cells and brain tissue at 60 min. The microglial cells and brain tissue were normalized by levels of total proteins detected by BCA. n = 3–5 per time point for each groups. Data are presented as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 2

Preparation and characterizations of the corona formed on NP and NP-αM in 0d-plasma or 7d-plasma. (A) Schematic illustration of the NP-corona complexes preparation setup: the NP and NP-αM were incubated with plasma from untreated C57 mice or animal treated with the nanoparticles for 7 days. (B and C) Morphology and particle size distribution of NP/0d-NP-corona/7d-NP-corona (B) and NP-αM/0d-NP-αM-corona/7d-NP-αM-corona (C) under TEM and DLS. Scale bar = 50 nm. (D) Particle size and PDI of the nanoparticles (n = 5). (E) Zeta potential of the nanoparticles (n = 5).

Figure 3

Cellular uptake of NP-corona complexes under different loading and incubation conditions in b.End3 and BV-2 cells. (A and C) Quantitative analysis of NP, NP-αM and NP-corona complexes in b.End3 (A) and BV-2 (C) cells. NP-corona complexes formed on NP or NP-αM in 0d-plasma or 7d-plasma were isolated as described in the Methods. All nanoparticles were labeled with cou6 and incubated with cells for 3 h at the concentration of 15 μg/mL. (B and D) Laser confocal imaging of NP, NP-αM and NP-corona complexes in b.End3 (B) and BV-2 (D) cells, Scale bar = 50 μm. Data are presented as the mean ± SD (n = 5). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 4

Blood–brain barrier permeability of the NP–corona complexes. (A) Schematic diagram of the in vitro BBB-Transwell model. (B) Laser confocal imaging of tight junction protein claudin-5 in the BBB model. Scale bar = 40 μm. (C) The TEER of b.End3 cells were measured at the culture time of 2, 4 and 6 days. (D) The percentage of NP-corona complexes transported across the BBB (n = 3). (E and F) Quantitative analysis (E) and laser confocal imaging (F) of the cellular uptake of NP-corona complexes in BV-2 cells cultured at the bottom of the BBB model, in which the nanoparticles firstly crossed the BBB and then got access to the microglia for 12 h, Scale bar = 40 μm. Green: cou6-Nano, Blue: Nucleus. Data are presented as the mean ± SD (n = 4). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 5

Proteomics analysis of the corona formed on NP or NP-αM in 0d-plasma or 7d-plasma. (A) Heatmap of relative abundance (log₂ scale) values for each corona protein between samples. (B) Venn diagram of the total amount of proteins in NP-corona complexes. (C) The light gray of the Venn diagram indicates the upregulated proteins of 0d-NP-αM-corona compared to 0d-NP-corona, and the dark gray indicates the upregulated proteins of 7d-NP-αM-corona compared to 7d-NP-corona (fold change > 1.5, P value < 0.05). Venn diagram reports the number of unique and common proteins. (D) The light gray of the Venn diagram indicates the upregulated proteins of 7d-NP-corona compared to 0d-NP-corona, and the dark gray indicates the upregulated proteins of 7d-NP-αM-corona compared to 0d-NP-αM-corona (fold change > 1.5). Venn diagram reports the number of unique and common proteins. (E) Heatmap of relative abundance (log₂ scale) values for the top 30 proteins (ordered by maximum fold change) among the common proteins in (C) are displayed. (F) Heatmap of relative abundance (log₂ scale) values for the top 60 proteins (ordered by maximum relative abundance values) among the common proteins in (D) are displayed. (G–J) GO analysis of the upregulated proteins (fold change > 1.5) of 7d-NP-corona compared to 0d-NP-corona (G and H) and 7d-NP-αM-corona compared to 0d-NP-αM-corona (I and J).

Figure 6

Uptake Mechanisms of NP-corona complexes under different loading and incubation conditions. NP-corona complexes formed on NP or NP-αM in 0d-plasma or 7d-plasma were isolated as described in the Methods. All NP-corona complexes were labeled with cou6 and incubated with cells for 3 h in DMEM at the concentration of 15 μg/mL. (A and C) b.End3 (A) and BV-2 (C) cells were exposed to 7d-NP-corona with or without the treatment of 5 μg/mL anti-CD16/32 or 10 m mol/L EDTA, and 0d-NP-corona as control. (B and D) b.End3 (B) and BV-2 (D) cells were exposed to 7d-NP-αM-corona with or without the treatment of 5 μg/mL anti-CD16/32 or 10 mmol/L EDTA, and 0d-NP-αM-corona as control. (E and G) b.End3 (E) and BV-2 (G) cells were exposed to 0d-NP-αM-corona with or without the treatment of 1 μg/mL anti-CD36 or 1 μg/mL anti-Rab5a, and 0d-NP-corona as control. (F and H) b.End3 (F) and BV-2 (H) cells were exposed to 7d-NP-αM-corona with or without the treatment of 1 μg/mL anti-CD36 or 1 μg/mL anti-Rab5a, and 7d-NP-corona as control. Data are presented as the mean ± SD (n = 5). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.