Stealth and Cationic Nanoliposomes as Drug Delivery Systems to Increase Andrographolide BBB Permeability

Abstract

(1) Background: Andrographolide (AG) is a natural compound effective for the treatment of inflammation-mediated neurodegenerative disorders. The aim of this investigation was the preparation of liposomes to enhance the penetration into the brain of AG, by modifying the surface of the liposomes by adding Tween 80 (LPs-AG) alone or in combination with Didecyldimethylammonium bromide (DDAB) (CLPs-AG). (2) Methods: LPs-AG and CLPs-AG were physically and chemically characterized. The ability of liposomes to increase the permeability of AG was evaluated by artificial membranes (PAMPA) and hCMEC/D3 cells. (3) Results: Based on obtained results in terms of size, homogeneity, ζ-potential and EE%. both liposomes are suitable for parenteral administration. The systems showed excellent stability during a month of storage as suspensions or freeze-dried products. Glucose resulted the best cryoprotectant agent. PAMPA and hCMEC/D3 transport studies revealed that LPs-AG and CLPs-AG increased the permeability of AG, about an order of magnitude, compared to free AG without alterations in cell viability. The caveolae-mediated endocytosis resulted the main mechanism of up-take for both formulations. The presence of positive charge increased the cellular internalization of nanoparticles. (4) Conclusions: This study shows that developed liposomes might be ideal candidates for brain delivery of AG.

Keywords: PAMPA; andrographolide; brain delivery; cationic liposomes; hCMEC/D3 cells; liposomes; surfactant.

Figure 1

TEM images of LPs-AG (a) and CLPs-AG (b) (scale 100 nm).

Figure 2

Particle size, polydispersity index (PdI) (a) and zeta-potential (b) of LPs-AG and CLPs-AG as dispersion after one-month storage at 4 °C. (Data displayed as mean ± SD; n = 3).

Figure 3

In vitro release profiles of LPs-AG and CLPs-AG in PBS. (each data point represents the average of three samples).

Figure 4

hCMEC/D3 cell viability evaluated by MTT assay (left panel) and cytotoxicity by LDH assay (right panel) when exposed for 2 h (a), 4 h (b) and 24 h (c) to AG (10 and 100 µM) or LPs-AG and CLPs-AG (0.0085 and 0.085 mg/mL). Data is expressed as percentage of control (EBM-2 medium) and Triton-X (TTX) which represent, respectively, the maximum cell viability and cell cytotoxicity. Values represent the mean ± SEM of at least three experiments performed in triplicate. * p < 0.05 and ** p < 0.01 vs. EBM-2 alone.

Figure 5

The apparent permeable coefficient of different liposomal formulations for different treatment time in the in vitro BBB model. (Data represent means ± S.D, n = 3).

Figure 6

Cellular uptake of LPs-6C and CLPs-6C by hCMEC/D3 cells after 2 h incubation at 37 °C. Images of nuclei stained with DAPI (blue), 6-Coumarin (green) and their overlay. Scale bar: 20 μm.

Figure 7

Effect of the temperature (4 °C) and different inhibitors on hCMEC/D3 cell internalization pathways of LPs-6C and CLPs-6C after 2 h incubation at 37 °C. (Data represent the mean ± standard deviation (n = 3). Bars represent the mean ± SD of at least 6 experiments ** p < 0.01 vs. corresponding liposome at 37 °C; ^# p < 0.05 CLPs-6C vs. LPs-6C. (ANOVA + Tukey’s test).