Cell-penetrating Peptide-coated Liposomes for Drug Delivery Across the Blood–Brain Barrier

Materials and Methods

Materials. R8 was synthesized by GL Biochem Ltd. (Shanghai, China). 1,2-Dioleoyl-3-trimethylammonium-propane chloride (DOTAP) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000] (PEG-DSPE) were purchased from Lipoid (Newark, NJ, USA). Egg phosphatidylcholine (ePC) was obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Oleic acid was obtained from Sigma (St. Louis, MO, USA). Modified Eagle's medium (MEM) and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT, USA). 4’,6-Diamidino-2-phenylindole (DAPI) was obtained from Molecular Probes Inc. (Eugene, OR, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). U87-MG, a glioblastoma cell line, was obtained from the American Type Culture Collection (Manassas, VA, USA).

Preparation and characterization of liposomes. R8-conjugated oleic acid (R8-OA) was synthesized according to a previously reported method (13). A series of liposomes with were prepared according to Table I (14): LP: Liposomes of ePC–cholesterol; PLP: liposomes of PEG-DSPE–LP; R8LP: R8-conjugated oleic acid liposomes of DOTAP–LP; R8PLP: R8-conjugated oleic acid liposomes of PLP. The composition of liposomes was adjusted to optimize uptake by U87-MG cells. Liposomes were prepared by an ethanol injection method and doxorubicin was actively loaded into liposomes by an ammonium sulfate gradient. Firstly, R8-OA, ePC, cholesterol, and PEG-DSPE were dissolved in ethanol at different ratios to form a mixture. This was then injected into ammonium sulfate (250 mM) at a volume ratio of 1:10 to form liposomes. Liposomes were dialyzed to remove the ammonium sulfate external to the liposomes. Finally, doxorubicin was added to initiate drug loading at final concentration of 1 mg/ml. The mean size and ζ-potential of liposomes were measured by Zetasizer Nano ZS 90 from Malvern Instruments, Ltd. (Malvern, U.K.). Surface morphology of liposomes was examined on a JEOL scanning electron microscope (SEM) (Tokyo, Japan). The encapsulation efficiency (EE%) of doxorubicin in liposomes was then evaluated.

The release of doxorubicin in vitro. A dialysis method was used to study the release of doxorubicin from liposomes. Briefly, liposomes were placed into dialysis tubes (molecular weight cut-off of 8,000-12,000 Da). The external solution for dialysis was phosphate-buffered saline (PBS). The dialysis was performed in the dark with stirring at 37°C. The external solution was periodically sampled and replaced with an equal volume of PBS.

The stability of R8PLP was evaluated in PBS with 10% FBS at 37°C. Liposomes were incubated for 0, 2, 4, 8, 12 or 24 h and measured the size and EE% of liposomes (15). The encapsulated doxorubicin (E_Dox) in R8PLP was separated from liposomes by Sephadex G-50 (Yuanye Biological Technology Co.,Ltd., Shanghai, China). The total doxorubicin (T_Dox) was released from liposomes by Triton X-100 (Yuanye Biological Technology Co.,Ltd., Shanghai, China). The EE% was calculated using the following equation:

The concentration of doxorubicin was quantified by Agilent 1100 high-performance liquid chromatography (Santa Clara, CA, USA) using a fluorescence detector at Ex/Em of 480/525 nm using acetonitrile/water (50:50) as the mobile phase.

Cellular uptake of liposomes in vitro. Laser confocal microscopy was used to qualitatively analyze U87-MG cellular uptake. U87-MG cells were seeded into glass-bottom culture dish at a density of 1×10⁵ cells/well and cultured for 24 h at 37°C in 5% CO₂ atmosphere. Liposomes were then added to the U87-MG cells, which were then incubated for 4 h. The cells were fixed in 4% paraformaldehyde for 10 mins, washed with PBS. The nucleus was stained with 2 μg/ml of DAPI for 10 min. Finally, cells were washed with PBS three times and observed under a confocal microscopy from Carl Zeiss (Oberkochen, Jena, Germany) on the red channel.

The cellular uptake of liposomes was further quantitatively measured by flow cytometry. U87-MG cells were plated into 6-well plates at the density of 1×10⁵ cells/well and cultured for 24 h at 37°C in 5% CO₂ atmosphere. Liposomes were then added to the U87-MG cells, which were incubated for 4 h. The cells were fixed with 4% paraformaldehyde for 10 mins and washed with PBS. Fluorescence intensity of cells was measured on a flow cytometer from Beckman Coulter (Brea, CA, USA).

Cytotoxicity assay. Briefly, 1×10⁴ cells per well were seeded into 96-well plates at 37°C and cultured for 24 h. Liposomes with doxorubicin concentration of 3.6 μM were added and the cells were incubated for 24, 48 and 72 h after treatment. Ten microliters of MTT (5 mg/ml) was added and the plates were incubated for 4 h. Formazan was dissolved in 150 μl dimethylsulfoxide and the absorbance at 490 nm measured by on a BioTek Synergy4 microplate reader (Winooski, VT, USA).

Animals. In order to determine whether R8-OA-modified liposomes were able to penetrate the BBB and increase doxorubicin accumulation in the brain, an in vivo study was carried out in mice (16, 17). Female KM mice (30±2 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal experiments were approved by the Animal Ethics Committee of the Jilin University (No. 201805010). Mice were separated into two groups of 21 each. Mice were fasted for 12 h before administering liposomes. Mice received a single dose of liposomes (3 mg/kg of doxorubicin) through tail vein injection. Mice were sacrificed and the brain was collected at 0.5, 1, 2, 4, 6, 8, or 12 h. Tissue samples were used for analysis of doxorubicin content.

Distribution of doxorubicin in the brain. Doxorubicin analysis in brain tissue was established by Waters Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) (Milford, MA, USA) using daunorubicin as internal standard (IS). The method was reported previously (18). Briefly, brain tissue was homogenized with two volumes of water, IS was added to 100 μl homogenization solution at a final concentration of 20 ng/ml. Samples were treated with 200 μl borate buffer (6% of w/v, pH 9.5) for 2 mins under vortex. Doxorubicin and IS were extracted from the mixture by 1 mI of chloroform/1-propanol (4:1, v/v) for 2 min with mixing and the organic phase was separated and dried by nitrogen. The residue was resuspended by the initial mobile phase and 10 μl of sample was injected into the UPLC-MS/MS system.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Characterization of R8-conjugated oleic acid liposomes of 1,2-dioleoyl-3-trimethylammonium-propane chloride–egg phosphatidylcholine–cholesterol–1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000] (R8PLP). The diameter and morphology of R8PLP were characterized. The size of R8PLP was measured in intensity-weighted Gaussian size distribution mode, the polydispersity index was 0.133. Scanning electron microscopy image was obtained in secondary electron imaging mode: 3.0 kV, ×30,000.

UPLC-MS/MS conditions: Source temperature 150°C, capillary voltage 3 kV, cone voltage 30 V, and desolvation temperature was set at 500°C. Doxorubicin was measured by quantitative ions of m/z 544.20→397.21 and m/z 528.18→321.19 for doxorubicin and IS, respectively.

Statistical analysis. The data are described as the mean±SD. The statistical significance of results was calculated by t-test. A p-value of less than 0.05 was accepted as indicating a significant difference.