Brain-targeted drug delivery by manipulating protein corona functions

Abstract

Protein corona presents a major obstacle to bench-to-bedside translation of targeted drug delivery systems, severely affecting targeting yields and directing unfavorable biodistribution. Corona-mediated targeting provides a new impetus for specific drug delivery by precisely manipulating interaction modes of functional plasma proteins on nano-surface. Here bio-inspired liposomes (SP-sLip) were developed by modifying liposomal surface with a short nontoxic peptide derived from Aβ_1-42 that specifically interacts with the lipid-binding domain of exchangeable apolipoproteins. SP-sLip absorb plasma apolipoproteins A1, E and J, consequently exposing receptor-binding domain of apolipoproteins to achieve brain-targeted delivery. Doxorubicin loaded SP-sLip (SP-sLip/DOX) show significant enhancement of brain distribution and anti-brain cancer effect in comparison to doxorubicin loaded plain liposomes. SP-sLip preserve functions of the absorbed human plasma ApoE, and the corona-mediated targeting strategy works in SP modified PLGA nanoparticles. The present study may pave a new avenue to facilitate clinical translation of targeted drug delivery systems.

Fig. 1

Binding kinetics and capacity of recombinant human ApoE (rhApoE) on liposomal surface. a, b rhApoE and the same amount of BSA mixture in 100 μL PBS was incubated with 100 μL sLip or SP-sLip for 0, 5, 30, 60, 120, and 240 min at 37 °C (rhApoE, 10 μg mL⁻¹; phospholipid, 5 mg mL⁻¹). Protein coronas were obtained by centrifugation at 14k RCF for 20 min, loaded to 4–20% SDS-PAGE gel for separation and stained with silver. The band intensity of absorbed BSA and rhAPOE was calculated by image J software and normalized by setting BSA intensity in the control panel as 100. c, d rhApoE (final concentration of 0, 1, 10, and 50 μg mL⁻¹) and the same amount of BSA mixture in 100 μL PBS was incubated with 100 μL sLip or SP-sLip for 1 h at 37 °C. Protein coronas were obtained as in a. The band intensity of absorbed rhApoE was calculated and normalized by setting that in the 50 μg mL⁻¹ rhApoE panel as 100. Data are means ± SD (n = 3), and analyzed with GraphPad Prism 6.0. **p < 0.01, ***p < 0.001 by student’s t-test

Fig. 2

Characterization of protein coronas in vitro and in vivo. a sLip or SP-sLip were incubated with mouse plasma for 1 h, and the protein coronas were isolated by centrifugation at 14k RCF, separated by SDS-PAGE and stained with silver. b The band intensity of red arrows (ascertained as ApoJ, ApoE, and ApoA1 by nano-LC-MS/MS) was calculated by image J software. The intensity of each apolipoprotein in the formed protein corona on sLip at 0 min was set as 1 for normalization. c, d In vivo absorption of ApoJ, ApoE, and ApoA1 on liposomal surface. DiI-loaded sLip or SP-sLip (100 μL, DiI 0.4 mg mL⁻¹, phospholipid 10 mg mL⁻¹) was injected into BALB/c mice via tail vein. Blood was sampled at 1 h after administration and protein coronas were isolated by centrifugation at 14k RCF. Western Blotting was applied to detect the absorbed ApoJ, ApoE, and ApoA1. Quantitative analysis of proteins was performed by Image J software and the average band intensity of each protein in the formed protein corona on sLip was set as 1 for normalization. Data are means ± SD (n = 3), and analyzed with GraphPad Prism 6.0. *p < 0.05, **p < 0.01, ***p < 0.001 by student’s t-test

Fig. 3

Characterization of ApoE functions in protein corona. a sLip or SP-sLip (100 μL, 10 mg mL⁻¹ lipid) were incubated with BSA (10 μg mL⁻¹) with or without rhApoE (10 μg mL⁻¹) for 1 h at 37 °C, then recombinant human LRP1 (rhLRP1) was added and incubated for another 1 h at 37 °C. Protein coronas were isolated by centrifugation at 14k RCF, separated by SDS-PAGE and stained with silver. b SP-PEG3400-DSPE dissolved in methanol (0.1 μg per well) was coated in 96-well ELISA plate. After dry in room temperature, it was rinsed by cold PBS for three times. BSA (3%) was used to block for 2 h at room temperature. After thrice PBST rinses, anti-SP antibody (rabbit IgG, containing 0.1% BSA) was added and incubated for 1 h at 37 °C. Different concentrations of free SP (from 0 to 100 μg mL⁻¹) or liposomes (sLip or SP-sLip, 0–5 mg mL⁻¹ lipid) with or without plasma were incubated for another 1 h at 37 °C. HRP-anti-rabbit IgG was applied to detect the primary antibody using TMB kit. c sLip or SP-sLip were incubated with plasma for 1 h at 37 °C, then I¹²⁵ radiolabeled rhLRP1 was added in the mixture for another 1 h incubation. The radioactivity of liposomes after three cycles centrifugation and rinses was counted for detecting the absorbed rhLRP1. d Microscopic observation of cellular uptake of DiI-loaded sLip (sLip/DiI) or SP-sLip (SP-sLip/DiI) by bEnd.3 cells. sLip/DiI or SP-sLip/DiI was preincubated with PBS, rhApoE (10 μg mL⁻¹) or mouse plasma for 1 h at 37 °C, then incubated with bEnd.3 cells for 4 h at 37 °C. Cellular uptake was detected by confocal laser scanning microscopy. Scale bar = 20 μm. e Quantitative analysis of DiI positive bEnd.3 cells by flow cytometry. Data are means ± SD (n = 3), and analyzed with GraphPad Prism 6.0. ***p < 0.001 by student’s t-test

Fig. 4

Biodistribution of sLip and SP-sLip in healthy BALB/c mice. a DiI-labeled sLip (sLip/DiI) and SP-sLip (SP-sLip/DiI) were injected via tail vein and brains were dissected and sectioned at 4 h after administration. Brain slices were stained with anti-CD31 antibody (green) and biodistribution of liposomes (red) in hippocampus (Hippo) and cortex were observed by confocal laser scanning microscopy. Scale bar = 20 μm. Doxorubicin distribution in brain (b) and other main organs (c) at 1 h, 4 h, and 24 h after intravenous injection of SP-sLip/DOX and sLip/DOX. Data are means ± SD (n = 3–5). *p < 0.05, **p < 0.01 by student’s t-test

Fig. 5

Biodistribution of sLip and SP-sLip in intracranial glioma. DiI-labeled sLip (sLip/DiI) and SP-sLip (SP-sLip/DiI) were injected via tail vein of nude mice bearing intracranial glioma (at day 16 after implantation). Mouse brains were dissected and sectioned at 4 h after administration. Brain slices were stained with anti-CD31 antibody (green) and biodistribution of liposomes (red) in normal brain tissues (labeled with Brain) and glioma region (labeled with Glioma) were observed by confocal laser scanning microscopy and quantified by Image Pro. Scale bar = 20 μm. Data are means ± SD (n = 3). ***p < 0.001 by student’s t-test

Fig. 6

Kaplan–Meier survival curve of nude mice bearing intracranial brain cancer cells after treatments. For mouse bearing U87 cells (a), saline (27 days, n = 12), DOX (31 days, n = 13), sLip/DOX (33 days, n = 13), and SP-sLip/DOX (50 days, n = 13) were intravenously administered at a total DOX dose of 10 mg kg⁻¹ (injections at day 7, 9, 11, 13, and 15 after U87 cells implantation). For mouse bearing D425 (b), saline (22 days, n = 8), DOX (23.5 days, n = 8), sLip/DOX (25 days, n = 7), and SP-sLip/DOX (29 days, n = 9) were intravenously administered at a total DOX dose of 12 mg kg⁻¹ (injections at day 3, 5, 7, 10, 12, and 14 days after D425 cells implantation). Data were plotted and the median survival time was calculated using GraphPad Prism 6.0 (Log-rank (Mantel–Cox) test)

Fig. 7

Evaluation of immunocompatibility of SP-sLip. a Immune response caused by sLip or SP-sLip in C57BL/6 mice. Absorbance in the ELISA plate versus serum dilution and antibody titer reported as log (EC50). mPEG2000-DSPE and SP-PEG3400-DSPE were used as antigens for sLip and SP-sLip, respectively. Data are means ± SD (n = 5). b Pharmacokinetic profile of DiI-labeled sLip and SP-sLip in SD rats over 24 h after intravenous injection. Data are means ± SD (n = 4) c PK parameters are calculated by DAS 2.0, and titration of IgG and IgM was calculated using GraphPad Prism 6.0. **p < 0.01 by student’s t-test

Fig. 8

Characterization of ApoE function in protein corona formed in human plasma. sLip and SP-sLip (a), or plain PLGA nanoparticles (PLGA NP) and SP-modified PLGA nanoparticles (SP-PLGA NP) (b) were incubated with human plasma for 1 h at 37 °C, then I¹²⁵ radiolabeled rhLRP1 was added in the mixture for another 1 h incubation. The radioactivity of liposomes and PLGA nanoparticles after three cycles centrifugation and rinses was counted for detecting the absorbed rhLRP1. Data are means ± SD (n = 4), **p < 0.01, ***p < 0.001 by student’s t-test