Targeting the brain with PEG-PLGA nanoparticles modified with phage-displayed peptides

Abstract

The relative impermeability of the blood-brain barrier (BBB) results from tight junctions and efflux transport systems limits drug delivery to the central nervous system (CNS), and thus severely restricts the therapy of many central nervous system diseases. In order to enhance the brain-specific drug delivery, we employed a 12-mer phage display peptide library to isolate peptides that could target the drug delivery system to the brain. A 12-amino-acid-peptide (denoted as Pep TGN) which was displayed by bacteriophage Clone 12-2 was finally selected by rounds of in vivo screening. Pep TGN was covalently conjugated onto the surface of poly (ethyleneglycol)-poly (lactic-co-glycolic acid) (PEG-PLGA) based nanoparticles (NPs). The cellular uptake of Pep TGN decorated nanoparticles was significantly higher than that of unmodified nanoparticles when incubated with bEnd.3 cells. Enhanced brain accumulation efficiency together with lower accumulation in liver and spleen was observed in the nude mice intravenously injected with Pep TGN conjugated nanoparticles compared with those injected with plain nanoparticles, showing powerful brain selectivity of Pep TGN. Coumarin 6 was used as a fluorescent probe for the evaluation of brain delivery properties. The brain Drug Targeting Index (DTI) of coumarin 6 incorporated in targeted nanoparticles was significantly higher than that of coumarin 6 incorporated in plain nanoparticles. In conclusion, the Pep TGN is a motif never been reported before and Pep TGN modified nanoparticles showed great potential in targeted drug delivery across the blood brain barrier.

Fig 1. (A) Four rounds of in vivo screening

Phages were injected i.v. into four groups of ICR mice. Rescued phages from the fourth round had a great augment (mean±S.D., n=3, *** p<0.005). (B) The time curve of brain/blood ratio of M13 phages titer. The accumulation of intact infectious phage particles in brains was analyzed by the ratio of phage particles in brain and in blood. The accumulation of phages in brain peaked at 24h. (mean±S.D., n=3). (C) Pep TGN competition with Clone 12-2. At the amount of 20μg, Pep TGN efficiently prevent the Clone 12-2 entering into the brain. (mean±S.D.), n=3 **p< 0.01, ***p<0.005).

Fig 2. Brain distribution of Bacteriophage in brain

Distribution of Clone 12-2 (A-D) and native M13 phage (E-H) in third ventricle (A,E), lateral ventricle (B,F), periventricular region of the third ventricle (C,G) and cortex (D,H) visualized 1h after i.v. administration in mouse caudal vein. Red: a secondary Cy3 conjugated Anti-mouse IgG; Blue: cell nuclei stained with 1μg/mL DAPI for 10min. The magnification bar represents 100μm.

Fig 3. Transmission electron micrographs of NP and TGN-NP

Different nanoparticles were negatively stained with phosphotungstic acid solution and observed under transmission electron micrographs: (A) NP; (B) TGN-NP (1:3); (C) TGN-NP (1:1). The bar is 200nm.

Fig 4. In vitro cytotoxicity

Cytotoxicity of NP, TGN-NP (1:3) and TGN-NP (1:1) incubated with bEnd.3 cells for (A) 4h, (B) 24h at 37°C. Cells without exposure to samples were used as control.

Fig 5. In vitro uptake of NP and TGN-NP

bEnd.3 cells uptake 50μg/mL NP at 37°C for 30min(A/B), 60min(C/D) and 120min(E/F) and 50μg/mL TGN-NP(1:1) for 30min(I/J), 60min(K/L) and 120min(M/N), respectively. bEnd.3 cells were incubated with free Pep TGN 1h before 50μg/mL NP (G/H) or 50μg/mL TGN-NP (O/P). Phase contrast photo were attached below each corresponding fluorescent photo. The bar is 25μm.

Fig 6. In vivo distribution of TGN-NP

(A) Optical in vivo imaging of nude mouse vein administrated (II) NP; (III) TGN-NP (1:1); (IV) TGN-NP (1:3) respectively. (I) blank nude mice as blank control. Images were taken 1h after injection. (B) Ex vivo imaging of NPs in main organs. Nude mice were tail intravenously injected with (II) NP; (III) TGN-NP (1:1) ; (IV) TGN-NP (1:3) respectively. (I) blank nude mice as blank control. Organs were harvested 2h after administration.

Fig 7. Concentrations-time curves of coumarin 6 in cerebrum

ICR mice were i.v. injected of coumarin-6-loaded NP, TGN-NP (1:3) and TGN-NP (1:1) at a dose of 30μg/kg in mice, respectively. Data represented the mean±S.D. n=4.